U.S. patent application number 10/480409 was filed with the patent office on 2004-12-09 for electronic detection of biological molecules using thin layers.
Invention is credited to Mathai, George T., Sandeep, Kunwar, Sobha M., Pisharody.
Application Number | 20040248282 10/480409 |
Document ID | / |
Family ID | 27404499 |
Filed Date | 2004-12-09 |
United States Patent
Application |
20040248282 |
Kind Code |
A1 |
Sobha M., Pisharody ; et
al. |
December 9, 2004 |
Electronic detection of biological molecules using thin layers
Abstract
This invention provides novel sensors that facilitate the
detection of essentially any analyte. In general, the biosensors of
this invention utilize a binding agent (e.g. biomolecule) to
specifically bind to one or more target analytes. In preferred
embodiments, the biomolecules spans a gap between two electrodes.
Binding of the target analyte changes conductivity of the sensor
thereby facilitating ready detection of the binding event and thus
detection and/or quantitation of the bound analyte.
Inventors: |
Sobha M., Pisharody; (Castro
Valley, CA) ; Sandeep, Kunwar; (Redwood City, CA)
; Mathai, George T.; (Castro Valley, CA) |
Correspondence
Address: |
FENWICK & WEST LLP
SILICON VALLEY CENTER
801 CALIFORNIA STREET
MOUNTAIN VIEW
CA
94041
US
|
Family ID: |
27404499 |
Appl. No.: |
10/480409 |
Filed: |
July 16, 2004 |
PCT Filed: |
June 10, 2002 |
PCT NO: |
PCT/US02/18319 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60297583 |
Jun 11, 2001 |
|
|
|
60378938 |
May 10, 2002 |
|
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Current U.S.
Class: |
435/287.2 ;
435/6.11 |
Current CPC
Class: |
G11C 13/0014 20130101;
F41H 11/12 20130101; G01N 27/3278 20130101; G11C 13/0019 20130101;
C12Q 1/003 20130101; C12Q 1/6825 20130101; G01N 33/5438 20130101;
B82Y 10/00 20130101 |
Class at
Publication: |
435/287.2 ;
435/006 |
International
Class: |
C12M 001/34; C12Q
001/68 |
Foreign Application Data
Date |
Code |
Application Number |
Oct 2, 2001 |
US |
09/970,087 |
Claims
What is claimed is:
1. A molecular sensing apparatus comprising: a substrate; an
insulator overlying said substrate; and one or more electrode
pairs, wherein a first electrode pair in said one or more electrode
pairs comprises: a spacer overlaying a first portion of said
insulator; a first electrode overlaying said spacer; and a second
electrode overlaying a second portion of said insulator, and
wherein said second electrode is adjacent to said spacer, wherein
said first electrode and said second electrode are separated by a
distance that would allow a biological macromolecule or biological
macromolecule/analyte complex to connect said first electrode to
said second electrode.
2. The molecular sensing apparatus of claim 1, wherein said
biological macromolecule or macromolecule/analyte complex connects
said first electrode and said second electrode in said first
electrode pair.
3. The molecular sensing apparatus of claim 2, wherein said
biological macromolecule is selected from the group consisting of a
nucleic acid, a protein, a polysaccharide, a lectin, a lipid, a
sugar, and a carbohydrate.
4. The molecular sensing apparatus of claim 2, wherein said
biological macromolecule is a nucleic acid.
5. The molecular sensing apparatus of claim 2, wherein said
biological macromolecule is functionalized with a chemical group
selected from the group consisting of a sulfate, a sulfhydryl, an
amine, an aldehyde, a carboxylic acid, a phosphate, a phosphonate,
an alkene, an alkyne, a hydroxyl, a bromine, an iodine, a chlorine,
a light-activatable group, and a group activatable by an electric
potential.
6. The molecular sensing apparatus of claim 1, wherein said spacer
comprises an insulator having a resistivity greater than 10.sup.-3
ohm-meters.
7. The molecular sensing apparatus of claim 1, wherein said spacer
comprises an insulator selected from the group consisting of
SiO.sub.2, TiO.sub.2, ZrO.sub.2, quartz, porcelain, ceramic,
polystyrene, TEFLON, and an insulating oxide or sulfide of a
transition metal in the periodic table of the elements.
8. The molecular sensing apparatus of claim 1, wherein said first
electrode and said second electrode are separated by a distance in
the range of 1 Angstrom to 10.sup.9 Angstroms.
9. The molecular sensing apparatus of claim 1, wherein said first
electrode and said second electrode are separated by a distance
less than 500 Angstroms.
10. The molecular sensing apparatus of claim 1, wherein at least
one of said first electrode and said second electrode has a
resistivity of less than 10.sup.-2 ohm-meters.
11. The molecular sensing apparatus of claim 1, wherein at least
one of said first electrode and said second electrode has a
resistivity of less than 10.sup.-3 ohm-meters.
12. The molecular sensing apparatus of claim 1, wherein said first
electrode and said second electrode comprise a material selected
from the group consisting of ruthenium, osmium, cobalt, rhodium,
rubidium, lithium, sodium, potassium, vanadium, cesium, beryllium,
magnesium, calcium, chromium, molybdenum, silicon, germanium,
aluminum, iridium, nickel, palladium, platinum, iron, copper,
titanium, tungsten, silver, gold, zinc, cadmium, indium tin oxide,
carbon, and carbon nanotube.
13. The molecular sensing apparatus of claim 1, wherein said first
electrode is functionalized with a chemical group that can be
derivatized or crosslinked.
14. The molecular sensing apparatus of claim 13, wherein said
chemical group is a sulfate, a sulfhydryl, an amine, an aldehyde, a
carboxylic acid, a phosphate, a phosphonate, an alkene, an alkyne,
a hydroxyl group, a bromine, an iodine, a chlorine, a
light-activatable group, or a group activatable by an electric
potential.
15. The molecular sensing apparatus of claim 1, wherein at least
one of said first electrode and said second electrode is coated
with a self-assembled monolayer (SAM).
16. The molecular sensing apparatus of claim 15, wherein said SAM
comprises a compound selected from the group consisting of an
alkanethiol, a phospholipid, a bola amphiphile, and an
oligo(phenylenevinylene).
17. The molecular sensing apparatus of claim 2, wherein the
biological macromolecule or macromolecular/analyte complex is
attached to the first electrode by a thiol group.
18. The molecular sensing apparatus of claim 2, wherein the
biological macromolecule or macromolecular/analyte complex is
attached to the first electrode by a phosphorothioate or a
phosphonate.
19. The molecular sensing apparatus of claim 2, wherein the
biological macromolecule or macromolecular/analyte complex is
attached to said first electrode by a linker.
20. The molecular sensing apparatus of claim 19, wherein said
linker is selected from the group consisting of DFDNB, DST, ABH,
ANB-NOS, EDC, NHS-ASA, and SIA.
21. The molecular sensing apparatus of claim 1, wherein said first
electrode comprises a surface with a shape selected from the group
consisting of convex, concave, textured, corrugated, patterned
uniformly, and randomly patterned.
22. The molecular sensing apparatus of claim 1, wherein the first
electrode in said first electrode pair has a first surface and the
second electrode in said first electrode pair has a second surface,
wherein the first surface is not coplanar to the second
surface.
23. The molecular sensing apparatus of claim 1, wherein said one or
more electrode pairs are at least 10 electrode pairs.
24. The molecular sensing apparatus of claim 1, wherein said one or
more electrode pairs are at least 10,000 electrode pairs.
25. The molecular sensing apparatus of claim 1, wherein said one or
more electrode pairs are 10.sup.2 to 10.sup.10 electrode pairs.
26. The molecular sensing apparatus of claim 1, wherein said one or
more electrode pairs are at least 10.sup.2 electrode pairs per
square centimeter of said insulating layer.
27. The molecular sensing apparatus of claim 1, wherein said one or
more electrode pairs are at least 1,000,000 electrode pairs per
square centimeter of said insulating layer.
28. The molecular sensing apparatus of claim 1, the apparatus
further comprising a measurement device electrically coupled to the
first electrode and to the second electrode of said first electrode
pair.
29. The molecular sensing apparatus of claim 28, wherein said
measurement device measures an electromagnetic property selected
from the group consisting of direct electric current, alternating
electric current, permitivity, resistivity, electron transfer,
electron tunneling, electron hopping, electron transport, electron
conductance, voltage, electrical impedance, signal loss,
dissipation factor, resistance, capacitance, inductance, magnetic
field, electrical potential, charge and magnetic potential.
30. The molecular sensing apparatus of claim 1 or 2, further
comprising an electrical circuit electrically coupled to the first
electrode and the second electrode.
31. The molecular sensing apparatus of claim 30, wherein said
electrical circuit comprises an electric signal gating system.
32. The molecular sensing apparatus of claim 31, wherein said
electric signal gating system comprises a CMOS gating system.
33. The molecular sensing apparatus of claim 1, wherein a first
biological molecule is attached to said first electrode in said
first electrode pair, and a second biological molecule is attached
to said second electrode in said first electrode pair; wherein said
first biological molecule and said second biological molecule are
the same.
34. The molecular sensing apparatus of claim 1, wherein a first
biological macromolecule or a first biological
macromolecule/analyte complex is attached to said first electrode
in said first electrode pair, and a second different biological
macromolecule or second different biological macromolecule/analyte
complex is attached to said second electrode in said first
electrode pair.
35. The molecular sensing apparatus of claim 1 or 2, further
comprising a computer electrically coupled to the first electrode
and the second electrode in said first electrode pair.
36. The molecular sensing apparatus of claim 1 or 2, wherein one of
the first electrode and the second electrode comprises a
semiconductor material.
37. The molecular sensing apparatus of claim 36, wherein said
semiconductor material has a resistivity ranging from 10.sup.-6
.OMEGA.-m to 10.sup.7 .OMEGA.-m.
38. The molecular sensing apparatus of claim 36, wherein the
semiconductor material is selected from the group consisting of
silicon, dense silicon carbide, boron carbide, Fe.sub.3O.sub.4,
germanium, silicon germanium, silicon carbide, tungsten carbide,
titanium carbide, indium phosphide, gallium nitride, gallium
phosphide, aluminum phosphide, aluminum arsenide, mercury cadmium
telluride, tellurium, selenium, ZnS, ZnO, ZnSe, CdS, ZnTe, GaSe,
CdSe, CdTe, GaAs, InP, GaSb, EnAs, Te, PbS, InSb, PbTe, PbSe, and
tungsten disulfide.
39. A method of making a molecular sensing apparatus comprising one
or more electrode pairs, said method comprising: (a) contacting a
first electrode and a second electrode in a first electrode pair in
said one or more electrode pairs with a first solution comprising a
biological macromolecule; (b) placing a charge on said first
electrode in said first electrode pair to attract said biological
macromolecule to said first electrode in said first electrode pair
so that said biological macromolecule attaches to said first
electrode to form a bound biological macromolecule; and (c) placing
a charge on said second electrode in said first electrode pair to
attract a portion of said bound biological macromolecule to said
second electrode in said first electrode pair so that said bound
biological macromolecule attaches to said second electrode, wherein
said first electrode and said second electrode in said first
electrode pair are separated by a spacer and are separated by a
distance that would allow said biological macromolecule or
biological macromolecule/analyte complex to connect said first
electrode to said second electrode.
40. The method of claim 39, wherein said first electrode, said
spacer, and said second electrode of an electrode pair in said one
or more electrode pairs do not form a sandwich configuration.
41. The method of claim 39 or 40, wherein said biological
macromolecule is selected from the group consisting of a nucleic
acid, a protein, a polysaccharide, a lectin, and a lipid.
42. The method of claim 39 or 40, wherein said biological
macromolecule is functionalized with a chemical group selected from
the group consisting of a sulfate, a sulfhydryl, an amine, an
aldehyde, a carboxylic acid, a phosphate, a phosphonate, an alkene,
an alkyne, a hydroxyl, a bromine, an iodine, a chlorine, a
light-activatable group, and a group activatable by an electric
potential.
43. The method of claim 39 or 40, wherein said biological
macromolecule is a nucleic acid.
44. The method of claim 39 or 40, wherein said spacer is an
insulator having a resistivity of greater than 10.sup.-3
.OMEGA.-m.
45. The method of claim 39 or 40, wherein said spacer comprises an
insulator selected from the group consisting of SiO.sub.2,
TiO.sub.2, ZrO.sub.2, porcelain, ceramic, quartz, high resistivity
plastic, and an insulating oxide or sulfide of the transition
metals in the periodic table of the elements.
46. The method of claim 39 or 40, wherein said first electrode and
said second electrode in said first electrode pair are separated by
a distance that is in a range from 10 Angstroms to 10.sup.5
Angstroms.
47. The method of claim 39 or 40, wherein said first electrode and
said second electrode in said first electrode pair are separated by
a distance that is less than 500 Angstroms.
48. The method of claim 39 or 40, wherein said first electrode and
said second electrode in said first electrode pair have a
resistivity of less than 10.sup.-3 .OMEGA.-m.
48. The method of claim 39 or 40, wherein at least one of said
first electrode and said second electrode in said first electrode
pair has a resistivity of less than 10.sup.-3 .OMEGA.-m.
49. The method of claim 39 or 40, wherein said first electrode and
said second electrode in said first electrode pair comprise a
material selected from the group consisting of ruthenium, osmium,
cobalt, rhodium, rubidium, lithium, sodium, potassium, vanadium,
cesium, beryllium, magnesium, calcium, chromium, molybdenum,
silicon, germanium, aluminum, iridium, nickel, palladium, platinum,
iron, copper, titanium, tungsten, silver, gold, zinc, cadmium,
indium tin oxide, carbon, and a carbon nanotube.
50. The method of claim 39 or 40, wherein said first electrode in
said first electrode pair is functionalized to bear a chemical
group capable of being further derivatized or crosslinked E prior
to said contacting step (a).
51. The method of claim 50, wherein said chemical group capable of
being further derivatized or crosslinked is selected from the group
consisting of a sulfate, a sulfhydryl, an amine, an aldehyde, a
carboxylic acid, a phosphate, a phosphonate, an alkene, an alkyne,
a hydroxyl group, a bromine, an iodine, a chlorine, a
light-activatable group, and a group activatable by an electric
potential.
52. The method of claim 39 or 40, wherein said biological
macromolecule is attached to said first electrode in said first
electrode pair by an electrically conductive linker.
53. The method of claim 52, wherein said linker is selected from
the group consisting of DFDNB, DST, ABH, ANB-NOS, EDC, NHS-ASA, and
SIA.
54. The method of claim 52, wherein said linker is
oligo(phenylenevinlyene- ).
55. The method of claim 39, wherein said one or more electrode
pairs are at least ten electrode pairs.
56. The method of claim 39, wherein said one or more electrode
pairs are at least 10,000 electrode pairs.
57. The method of claim 39, wherein said one or more electrode
pairs are 10.sup.2 to 10.sup.10 electrode pairs.
58. The method of claim 39, further comprising: (d) contacting a
first electrode and a second electrode in a second electrode pair
in said one or more electrode pairs with a second solution
comprising a second biological macromolecule; (e) placing a charge
on a first electrode in said second electrode pair to attract said
second biological macromolecule to said first electrode in said
second electrode pair so that said second biological macromolecule
attaches to said first electrode in said second electron pair to
form an attached second biological macromolecule; and (f) placing a
charge on said second electrode in said second electrode pair to
attract a portion of said attached second biological macromolecule
to said second electrode in said second electrode pair so that said
second biological macromolecule attaches to said second electrode
in said second electrode pair.
59. The method of claim 58, wherein said first solution and said
second solution are the same.
60. The method of claim 58, wherein said first solution and said
second solution are different.
61. The method of claim 58, wherein said first biological molecule
and said second biological molecule are the same.
62. The method of claim 58, wherein said first biological molecule
and said second biological molecule are the different.
63. The method of claim 39 or 40, wherein at least one of said
first electrode and said second electrode in said first electrode
pair comprises a semiconductor material.
64. The method of claim 63, wherein the semiconductor material has
a resistivity of less than 10.sup.-3 .OMEGA.-m.
65. The method of claim 63, wherein the semiconductor material is
selected from the group consisting of silicon, dense silicon
carbide, boron carbide, Fe.sub.3O.sub.4, germanium, silicon
germanium, silicon carbide, tungsten carbide, titanium carbide,
indium phosphide, gallium nitride, gallium phosphide, aluminum
phosphide, aluminum arsenide, mercury cadmium telluride, tellurium,
selenium, ZnS, ZnO, ZnSe, CdS, ZnTe, GaSe, CdSe, CdTe, GaAs, InP,
GaSb, InAs, Te, PbS, InSb, PbTe, PbSe, and tungsten disulfide.
66. The method of claim 39 wherein: said spacer overlays a first
portion of a substrate; said first electrode of said first
electrode pair overlays said spacer; and said second electrode of
said first electrode pair overlays a second portion of said
substrate, and wherein said second electrode is adjacent to said
spacer.
67. The method of claim 39 wherein there is an insulating layer
overlaying a substrate and said spacer overlays a first portion of
said insulator layer; said first electrode of said first electrode
pair overlays said spacer; and said second electrode of said first
electrode pair overlays a second portion of said insulator layer,
and wherein said second electrode is adjacent to said spacer.
68. The method of claim 39 wherein said first electrode pair is in
a spacer, and wherein said first electrode protrudes from said
substrate thereby forming a channel with walls formed, in part, by
said first electrode and said second electrode.
69. The method of claim 39 wherein said first electrode pair is in
a substrate and wherein a portion of said substrate is removed
between said first electrode and said second electrode in said
first electrode pair thereby forming a channel with walls formed by
said first electrode and said second electrode, and wherein there
is a biasing electrode in said channel.
70. A molecular sensing apparatus comprising one or more electrode
pairs in a substrate, wherein a first electrode pair in said one or
more electrode pairs comprises a first electrode and a second
electrode, wherein a portion of the substrate is removed between
said first electrode and said second electrode thereby forming a
channel within said substrate with walls formed by said first
electrode and said second electrode in said first electrode
pair.
71. The molecular sensing apparatus of claim 70 wherein said first
electrode and said second electrode in said first electrode pair
are separated by a distance that would allow a biological
macromolecule to connect said first electrode and said second
electrode.
72. The molecular sensing apparatus of claim 70 wherein a
biological macromolecule connects said first electrode and said
second electrode.
73. The molecular sensing apparatus of claim 72 wherein said
biological macromolecule is selected from the group consisting of a
nucleic acid, a protein, a polysaccharide, a lectin, a lipid, a
sugar, and a carbohydrate.
74. The molecular sensing apparatus of claim 72 wherein said
biological macromolecule is a nucleic acid.
75. The molecular sensing apparatus of claim 74 wherein said
nucleic acid is DNA or mRNA.
76. The molecular sensing apparatus of claim 71 wherein said
distance is in the range of 1 Angstrom to 10.sup.9 Angstroms.
77. The molecular sensing apparatus of claim 71 wherein said
distance is less than 500 Angstroms.
78. The molecular sensing apparatus of claim 70 wherein at least
one of said first electrode and said second electrode has a
resistivity of less than 10.sup.-2 ohm-meters.
79. The molecular sensing apparatus of claim 70 wherein at least
one of said first electrode and said second electrode has a
resistivity of less than 10.sup.-3 ohm-meters.
80. The molecular sensing apparatus of claim 79 wherein said first
electrode and said second electrode comprise a material selected
from the group consisting of ruthenium, osmium, cobalt, rhodium,
rubidium, lithium, sodium, potassium, vanadium, cesium, beryllium,
magnesium, calcium, chromium, molybdenum, silicon, germanium,
aluminum, iridium, nickel, palladium, platinum, iron, copper,
titanium, tungsten, silver, gold, zinc, cadmium, indium tin oxide,
carbon, and carbon nanotube.
81. The molecular sensing apparatus of claim 80 wherein said first
electrode is functionalized with a chemical group that can be
derivatized or crosslinked.
82. The molecular sensing apparatus of claim 81 wherein said
chemical group is a sulfate, a sulfhydryl, an amine, an aldehyde, a
carboxylic acid, a phosphate, a phosphonate, an alkene, an alkyne,
a hydroxyl, a bromine, an iodine, a chlorine, a light-activatable
group, or a group activatable by an electric potential.
83. The molecular sensing apparatus of claim 70 wherein at least
one of said first electrode and said second electrode is coated
with a self-assembled monolayer (SAM).
84. The molecular sensing apparatus of claim 83 wherein said SAM
comprises a compound selected from the group consisting of an
alkanethiol, a phospholipid, a bola amphiphile, and an
oligo(phenylenevinylene).
85. The molecular sensing apparatus of claim 72 wherein the
biological macromolecule is attached to the first electrode or the
second electrode by a thiol group.
86. The molecular sensing apparatus of claim 72 wherein the
biological macromolecule is attached to the first electrode or the
second electrode by a phosphorothioate or a phosphonate.
87. The molecular sensing apparatus of claim 72 wherein the
biological macromolecule is attached to said first electrode or the
second electrode by a linker.
88. The molecular sensing apparatus of claim 87 wherein said linker
is selected from the group consisting of DFDNB, DST, ABH, ANB-NOS,
EDC, NHS-ASA, and SIA.
89. The molecular sensing apparatus of claim 70 wherein the first
electrode has a first surface and the second electrode has a second
surface, and wherein the first surface is not coplanar to the
second surface.
90. The molecular sensing apparatus of claim 70 wherein said one or
more electrode pairs are at least 10 electrode pairs.
91. The molecular sensing apparatus of claim 70 wherein said one or
more electrode pairs are at least 10,000 electrode pairs.
92. The molecular sensing apparatus of claim 70 wherein said one or
more electrode pairs are at least 1,000,000 electrode pairs.
93. The molecular sensing apparatus of claim 70 wherein said one or
more electrode pairs are at least 10.sup.2 electrode pairs per
square centimeter of said substrate.
94. The molecular sensing apparatus of claim 70 wherein said one or
more electrode pairs are at least 1,000,000 electrode pairs per
square centimeter of said substrate.
95. The molecular sensing apparatus of claim 70 the apparatus
further comprising a measurement device electrically coupled to the
first electrode and to the second electrode of at least one
electrode pair in said one or more electrode pairs.
96. The molecular sensing apparatus of claim 95 wherein said
measurement device measures an electromagnetic property selected
from the group consisting of direct electric current, alternating
electric current, permitivity, resistivity, electron transfer,
electron tunneling, electron hopping, electron transport, electron
conductance, voltage, electrical impedance, signal loss,
dissipation factor, resistance, capacitance, inductance, magnetic
field, electrical potential, charge and magnetic potential.
97. The molecular sensing apparatus of claim 70 further comprising
an electrical circuit electrically coupled to the first electrode
and the second electrode.
98. The molecular sensing apparatus of claim 97 wherein said
electrical circuit comprises an electric signal gating system.
99. The molecular sensing apparatus of claim 98 wherein said
electric signal gating system comprises a CMOS gating system.
100. The molecular sensing apparatus of claim 70 wherein a nucleic
acid connects said first electrode and said second electrode.
101. The molecular sensing apparatus of claim 70 wherein a first
biological macromolecule is attached to said first electrode in
said first electrode pair, and a different second biological
macromolecule is attached to said second electrode in said first
electrode pair.
102. The molecular sensing apparatus of claim 70 further comprising
a computer electrically coupled to said first electrode and said
second electrode.
103. The molecular sensing apparatus of claim 70 wherein at least
one of the first electrode and the second electrode comprises a
semiconductor material.
104. The molecular sensing apparatus of claim 103 wherein said
semiconductor material has a resistivity of less than 10.sup.-3
.OMEGA.-m.
105. The molecular sensing apparatus of claim 104 wherein the
semiconductor material is selected from the group consisting of
silicon, dense silicon carbide, boron carbide, Fe.sub.3O.sub.4,
germanium, silicon germanium, silicon carbide, tungsten carbide,
titanium carbide, indium phosphide, gallium nitride, gallium
phosphide, aluminum phosphide, aluminum arsenide, mercury cadmium
telluride, tellurium, selenium, ZnS, ZnO, ZnSe, CdS, ZnTe, GaSe,
CdSe, CdTe, GaAs, InP, GaSb, EnAs, Te, PbS, InSb, PbTe, PbSe, and
tungsten disulfide.
106. A molecular sensing apparatus comprising: a substrate; and one
or more electrode pairs, wherein a first electrode pair in said one
or more electrode pairs comprises: a spacer overlaying a first
portion of said substrate; a first electrode overlaying said
spacer; and a second electrode overlaying a second portion of said
substrate, and wherein said second electrode is adjacent to said
spacer, wherein said first electrode and said second electrode are
separated by a distance that would allow a biological macromolecule
or biological macromolecule/analyte complex to connect said first
electrode to said second electrode.
107. The molecular sensing apparatus of claim 106, wherein said
biological macromolecule or macromolecule/analyte complex connects
said first electrode and said second electrode in said first
electrode pair.
108. The molecular sensing apparatus of claim 107, wherein said
biological macromolecule is selected from the group consisting of a
nucleic acid, a protein, a polysaccharide, a lectin, a lipid, a
sugar, and a carbohydrate.
109. The molecular sensing apparatus of claim 107, wherein said
biological macromolecule is a nucleic acid.
110. The molecular sensing apparatus of claim 107, wherein said
biological macromolecule is functionalized with a chemical group
selected from the group consisting of a sulfate, a sulfhydryl, an
amine, an aldehyde, a carboxylic acid, a phosphate, a phosphonate,
an alkene, an alkyne, a hydroxyl, a bromine, an iodine, a chlorine,
a light-activatable group, and a group activatable by an electric
potential.
111. The molecular sensing apparatus of claim 106, wherein said
first electrode and said second electrode are separated by a
distance in the range of 1 Angstrom to 10.sup.9 Angstroms.
112. The molecular sensing apparatus of claim 106, wherein said
first electrode and said second electrode are separated by a
distance less than 500 Angstroms.
113. The molecular sensing apparatus of claim 106, wherein at least
one of said first electrode and said second electrode has a
resistivity of less than 10.sup.-2 ohm-meters.
114. The molecular sensing apparatus of claim 106 wherein at least
one said first electrode and said second electrode has a
resistivity of less than 10.sup.-3 ohm-meters.
115. The molecular sensing apparatus of claim 106, wherein said
first electrode and said second electrode comprise a material
selected from the group consisting of ruthenium, osmium, cobalt,
rhodium, rubidium, lithium, sodium, potassium, vanadium, cesium,
beryllium, magnesium, calcium, chromium, molybdenum, silicon,
germanium, aluminum, iridium, nickel, palladium, platinum, iron,
copper, titanium, tungsten, silver, gold, zinc, cadmium, indium tin
oxide, carbon, and carbon nanotube.
116. The molecular sensing apparatus of claim 1, wherein said first
electrode is functionalized with a chemical group that can be
derivatized or crosslinked.
117. The molecular sensing apparatus of claim 13, wherein said
chemical group is a sulfate, a sulfhydryl, an amine, an aldehyde, a
carboxylic acid, a phosphate, a phosphonate, an alkene, an alkyne,
a hydroxyl group, a bromine, an iodine, a chlorine, a
light-activatable group, or a group activatable by an electric
potential.
118. The molecular sensing apparatus of claim 106, wherein at least
one of said first electrode and said second electrode is coated
with a self-assembled monolayer (SAM).
119. The molecular sensing apparatus of claim 118, wherein said SAM
comprises a compound selected from the group consisting of an
alkanethiol, a phospholipid, a bola amphiphile, and an
oligo(phenylenevinylene).
120. The molecular sensing apparatus of claim 107, wherein the
biological macromolecule or macromolecular/analyte complex is
attached to the first electrode by a thiol group.
121. The molecular sensing apparatus of claim 107, wherein the
biological macromolecule or macromolecular/analyte complex is
attached to the first electrode by a phosphorothioate or a
phosphonate.
122. The molecular sensing apparatus of claim 107, wherein the
biological macromolecule or macromolecular/analyte complex is
attached to said first electrode by a linker.
123. The molecular sensing apparatus of claim 122, wherein said
linker is selected from the group consisting of DFDNB, DST, ABH,
ANB-NOS, EDC, NHS-ASA, and SIA.
124. A molecular sensing apparatus comprising: one or more
electrode pairs, wherein at least one electrode pair in said one or
more electrode pairs comprises: a first electrode; a second
electrode; and an insulator between said first electrode and said
second electrode, wherein a channel is formed between said first
electrode and said second electrode; and wherein said first
electrode and said second electrode are separated by a distance
that would allow a biological macromolecule or biological
macromolecule/analyte complex to connect said first electrode to
said second electrode.
125. The molecular sensing apparatus of claim 124, wherein said
insulator has a resistivity greater than 10.sup.-3 ohm-meters.
126. The molecular sensing apparatus of claim 124, wherein said
insulator is selected from the group consisting of SiO.sub.2,
TiO.sub.2, ZrO.sub.2, quartz, porcelain, ceramic, polystyrene,
TEFLON, and an insulating oxide or sulfide of a transition metal in
the periodic table of the elements.
127. The molecular sensing apparatus of claim 124, wherein said
first electrode and said second electrode are separated by a
distance in the range of 10 Angstroms to 10.sup.5 Angstroms.
128. The molecular sensing apparatus of claim 124, wherein said
first electrode and said second electrode are separated by a
distance less than 500 Angstroms.
129. The molecular sensing apparatus of claim 124, wherein at least
one of said first electrode and said second electrode has a
resistivity of less than 10.sup.-2 ohm-meters.
130. The molecular sensing apparatus of claim 124, wherein at least
one of said first electrode and said second electrode has a
resistivity of less than 10.sup.-3 ohm-meters.
131. The molecular sensing apparatus of claim 124, wherein said
first electrode and said second electrode each comprises a material
selected from the group consisting of ruthenium, osmium, cobalt,
rhodium, rubidium, lithium, sodium, potassium, vanadium, cesium,
beryllium, magnesium, calcium, chromium, molybdenum, silicon,
germanium, aluminum, iridium, nickel, palladium, platinum, iron,
copper, titanium, tungsten, silver, gold, zinc, cadmium, indium tin
oxide, carbon, and carbon nanotube.
132. The molecular sensing apparatus of claim 124, wherein said
first electrode is functionalized with a chemical group that can be
derivatized or crosslinked.
133. The molecular sensing apparatus of claim 12, wherein said
chemical group is selected from the group consisting of a sulfate,
a sulfhydryl, an amine, an aldehyde, a carboxylic acid, a
phosphate, a phosphonate, an alkene, an alkyne, a hydroxyl, a
bromine, an iodine, a chlorine, a light-activatable group, and a
group activatable by an electric potential.
134. The molecular sensing apparatus of claim 124, wherein at least
one of said first electrode and said second electrode is coated
with a self-assembled monolayer (SAM).
135. The molecular sensing apparatus of claim 134, wherein said SAM
comprises a compound selected from the group consisting of an
alkanethiol, a phospholipid, a bola amphiphile, and an
oligophenylenevinylene).
136. The molecular sensing apparatus of claim 124, further
comprising a substrate that supports the first electrode and the
second electrode, wherein the first electrode and the second
electrode are integrated with the substrate.
137. The molecular sensing apparatus of claim 124 wherein said
first electrode comprises a surface with a shape selected from the
group consisting of convex, concave, textured, corrugated,
patterned uniformly, and randomly patterned.
138. The molecular sensing apparatus of claim 124 wherein said
first electrode and said second electrode are oriented in a
formation selected from the group consisting of annular, planar,
and orthogonal.
139. The molecular sensing apparatus of claim 124, wherein the
first electrode has a first surface and said second electrode has a
second surface, wherein the first surface is not coplanar to the
second surface.
140. The molecular sensing apparatus of claim 124, wherein said one
or more electrode pairs are at least 10 electrode pairs.
141. The molecular sensing apparatus of claim 124, wherein said one
or more electrode pairs are at least 10,000 electrode pairs.
142. The molecular sensing apparatus of claim 124, wherein said one
or more electrode pairs are at least 1,000,000 electrode pairs.
143. The molecular sensing apparatus of claim 124, the molecular
sensing apparatus further comprising a measurement device
electrically coupled to said first electrode and to said second
electrode.
144. The molecular sensing apparatus of claim 143, wherein said
measurement device measures an electromagnetic property selected
from the group consisting of direct electric current, alternating
electric current, permitivity, resistivity, electron transfer,
electron tunneling, electron hopping, electron transport, electron
conductance, voltage, electrical impedance, signal loss,
dissipation factor, resistance, capacitance, inductance, magnetic
field, electrical potential, charge and magnetic potential.
145. The molecular sensing apparatus of claim 124, further
comprising an electrical circuit electrically coupled to the first
electrode and the second electrode.
146. The molecular sensing apparatus of claim 145, wherein said
electrical circuit comprises an electric signal gating system.
147. The molecular sensing apparatus of claim 146, wherein said
electric signal gating system comprises a CMOS gating system.
148. The molecular sensing apparatus of claim 124, further
comprising a computer electrically coupled to the first electrode
and the second electrode.
149. The molecular sensing apparatus of claim 124, wherein at least
one of the first electrode and the second electrode comprises a
semiconductor material.
150. The molecular sensing apparatus of claim 149, wherein said
semiconductor material has a resistivity ranging from 10.sup.-6
.OMEGA.-m to 10.sup.7 .OMEGA.-m.
151. The molecular sensing apparatus of claim 149, wherein the
semiconductor material is selected from the group consisting of
silicon, dense silicon carbide, boron carbide, Fe.sub.3O.sub.4,
germanium, silicon germanium, silicon carbide, tungsten carbide,
titanium carbide, indium phosphide, gallium nitride, gallium
phosphide, aluminum phosphide, aluminum arsenide, mercury cadmium
telluride, tellurium, selenium, ZnS, ZnO, ZnSe, CdS, ZnTe, GaSe,
CdSe, CdTe, GaAs, InP, GaSb, EnAs, Te, PbS, InSb, PbTe; PbSe, and
tungsten disulfide.
152. A molecular sensing apparatus comprising: a substrate; one or
more electrode pairs, wherein a first electrode pair in said one or
more electrode pairs comprises: a spacer on a first portion of said
substrate; a first electrode on said spacer; and a second electrode
on a second portion of said substrate, and wherein said first
electrode and said second electrode are separated by a channel,
formed by said spacer and said second electrode, and wherein said
first electrode and said second electrode are separated by a
distance that would allow a biological macromolecule or biological
macromolecule/analyte complex to connect said first electrode to
said second electrode.
153. A molecular sensing apparatus comprising: a substrate; a
spacer on said substrate; one or more electrode pairs, wherein a
first electrode pair in said one or more electrode pairs comprises:
a first electrode on a first portion of said spacer; a channel
formed in a second portion of said spacer; and a second electrode
on the bottom of said channel, and wherein said first electrode and
said second electrode are separated by a distance that would allow
a biological macromolecule or biological macromolecule/analyte
complex to connect said first electrode to said second
electrode.
154. A method of detecting an analyte using the molecular sensing
apparatus of any of claims 1, 70, 106, 124, 152, or 153, the method
comprising: (i) attaching a biological macromolecule to said first
electrode; (ii) contacting the biological macromolecule with a
sample potentially comprising said analyte such that any analyte in
said sample binds to said biological macromolecule and forms a
macromolecule/analyte complex; (iii) placing a charge on said
second electrode to attract a portion of any first macromolecule
analyte complex to said second electrode thereby forming a
connection between said first electrode and said second electrode;
and (iv) detecting any said connection between said first electrode
and said second electrode.
155. A method of detecting an analyte using the molecular sensing
apparatus of any of claims 1, 70, 106, 124, 152, or 153, the method
comprising: (i) attaching a first biological macromolecule to said
first electrode; (ii) attaching a second biological macromolecule
to said second electrode; (iii) contacting said analyte with the
first biological macromolecule and the second biological
macromolecule thereby forming a macromolecule/analyte complex that
connects said first electrode and said second electrode in said
first electrode pair; and (iv) detecting the connection between
said first electrode and said second electrode in said first
electrode pair.
156. A method of detecting an analyte using the molecular sensing
apparatus of any of claims 1, 70, 106, 124, 152, or 153, the method
comprising: (i) detecting an electrical connection between said
first electrode and said second electrode when a biological
macromolecule forms a connection between said first electrode and
said second electrode; (ii) contacting the biological macromolecule
with said analyte whereby said analyte binds to said biological
macromolecule thereby forming a macromolecule/analyte complex; and
(iii) detecting a difference in the electrical connection between
said first electrode and said second electrode.
157. A method of detecting an analyte using the molecular sensing
apparatus of claim 2, the method comprising: (i) contacting said
biological macromolecule with a solution that potentially comprises
said analyte; and (ii) detecting a change in voltage or current
that arises between said first electrode and said second electrode
as a result of the formation of a macromolecule/analyte complex
between said analyte and said biological macromolecule.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation-in-part of U.S. Ser. No.
09/970,087, filed on Oct. 2, 2001, which claims priority to and
benefit of U.S. Ser. No. 60/297,583, filed on Jun. 11, 2001, both
of which are incorporated herein by reference in their entirety for
all purposes.
STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED
RESEARCH AND DEVELOPMENT
[0002] [Not Applicable]
FIELD OF THE INVENTION
[0003] This invention pertains to a biosensor for detecting and/or
quantifying analytes. More particularly, this invention pertains to
a biosensor based on a detection element that is a single
macromolecule spanning two electrodes.
BACKGROUND OF THE INVENTION
[0004] Biosensors are devices that can detect and/or quantify
analytes using known interactions between a targeted analyte and a
binding agent that is typically a biological macromolecule such as
an enzyme, receptor, nucleic acid, protein, lectin, or antibody.
Biosensors have applications in virtually all areas of human
endeavor. For example, biosensors have utility in fields as diverse
as blood glucose monitoring for diabetics, the recognition of
poisonous gas and/or explosives, the detection of chemicals
commonly associated with spoiled or contaminated food, genetic
screening, environmental testing, and the like.
[0005] Biosensors are commonly categorized according to two
features, namely, the type of macromolecule utilized in the device
and the means for detecting the contact between the binding agent
and the targeted analyte. Major classes of biosensors include
enzyme (or catalytic) biosensors, immunosensors and DNA
biosensors.
[0006] Enzyme (or catalytic) biosensors typically utilize one or
more enzymes as the macromolecule and take advantage of the
complimentary shape of the selected enzyme and the targeted
analyte. Enzymes are proteins that perform most of the catalytic
work in biological systems and are known for highly specific
catalysis. The shape and reactivity of a given enzyme limits its
catalytic activity to a very small number of possible substrates.
Enzyme biosensors rely on the specific chemical changes related to
the enzyme/analyte interaction as the means for recognizing contact
with the targeted analyte. For example, upon interaction with an
analyte, an enzyme biosensor may generate electrons, a colored
chromophore or a change in pH as the result of the relevant
enzymatic reaction. Alternatively, upon interaction with an
analyte, an enzyme biosensor may cause a change in a fluorescent or
chemiluminescent signal that can be recorded by an appropriate
detection system.
[0007] Immunosensors utilize antibodies as binding agents.
Antibodies are protein molecules that generally do not perform
catalytic reactions, but specifically bind to particular "target"
molecules (antigens). Antibodies are quite specific in their
interactions and, unlike most enzymes, they are capable of
recognizing and selectively binding to very large bodies such as
single cells. Thus, in addition to detection of small analytes,
antibody-based biosensors allow for the identification of certain
pathogens such as dangerous bacterial strains.
[0008] DNA biosensors typically utilize the complimentary nature of
the DNA or RNA double-strands and are designed for the specific
detection of particular nucleic acids. A DNA biosensor sensor
generally uses a single-stranded DNA as the binding agent. The
nucleic acid material in a given test sample is placed into contact
with the binding agent under conditions where the biosensor DNA and
the target nucleic acid analyte can form a hybrid duplex. If a
nucleic acid in the test sample is complementary to a nucleic acid
used in the biosensor, the two interact/bind. The interaction can
be monitored by various means such as a change in mass at the
sensor surface or the presence of a fluorescent or radioactive
signal. In alternative arrangements, the target nucleic acid(s) are
bound to the sensor and contacted with labeled probes to allow for
identification of the sequence(s) of interest.
[0009] While the potential utility for biosensors is great and
while hundreds of biosensors have been described in patents and in
the literature, actual commercial use of biosensors remains
limited. Aspects of biosensors that have limited their commercial
acceptance include a lack the sensitivity and/or speed of detection
necessary to accomplish certain tasks, problems with long term
stability, difficulty miniaturizing the sensor, and the like. In
addition, a number of biosensors must be pre-treated with salts
and/or enzyme cofactors, a practice that is inefficient and
bothersome.
SUMMARY OF THE INVENTION
[0010] This invention pertains to the development of a novel
molecular sensing apparatus (biosensor) and to methods of use
thereof. In preferred embodiments, the particular, the sensing
apparatus comprises a first electrode, a second electrode, an
insulator between the first and second electrode; and a binding
agent (e.g. a biological macromolecule) connecting the first
electrode to the second electrode. In particularly preferred
embodiments, the binding agent is attached to the electrode in a
manner that permits charge to flow from the electrode to the
binding agent or from the binding agent to the electrode. Preferred
binding agents include, but are not limited to biological
macromolecules (e.g. a nucleic acid, a protein, a polysaccharide, a
lectin, a lipid, etc.) with a nucleic acid being most preferred.
While the nucleic acid can be essentially any length preferred
nucleic acids range in length from about 5 to about 5,000
nucleotides, more preferably from about 8 to about 1,000 or 500
nucleotides, still more preferably from about 10 to about 300
nucleotides, and most preferably from about 15, 20, 25, 30, or 50
nucleotides to about 100 or 150 nucleotides in length. Typically
the nucleic acid is of sufficient length to specifically hybridize
to a target nucleic acid in a complex population of nucleic acids
(e.g. total genomic DNA) under stringent conditions.
[0011] In preferred embodiments, the biological macromolecule is
functionalized with a chemical group thereby facilitating the
attachment of the macromolecule to the electrode(s). Preferred
chemical groups include, but are not limited to a sulfate, a
sulfhydryl, an amine, an aldehyde, a carboxylic acid, a phosphate,
a phosphonate, an alkene, an alkyne, a hydroxyl group, a bromine,
an iodine, a chlorine, a light-activatable (labile) group, a group
activatable by an electric potential, and the like. In certain
embodiments, the biological macromolecule is functionalized with a
second biological macromolecule (e.g. a receptor, a receptor
ligand, an antibody, an epitope, a nucleic acid, a lectin, a sugar,
and the like). In preferred embodiments, however, such second
biological macromolecules exclude nucleic acids.
[0012] Preferred insulators are insulators having a resistivity
greater than about 10.sup.-3 ohm-meters, more preferably greater
than about 10.sup.-2 ohm-meters, and most preferably greater than
about 10.sup.-1, 1, or 10 ohm-meters. Suitable insulators include,
but are not limited to SiO.sub.2, TiO.sub.2, ZrO.sub.2, quartz,
porcelain, ceramic, polystyrene, Teflon (other high-resistivity
plastics), an insulating oxide or sulfide of a transition metal in
the periodic table of the elements, and the like.
[0013] In certain preferred embodiments, the first electrode and
the second electrode are separated by a distance in the range of 1
to 10.sup.9 Angstroms. Typically the first electrode and the second
electrode are separated by a distance less than about 300
Angstroms, preferably less than about 1.50 Angstroms, more
preferably less than about 500, preferably less than about 250,
more preferably less than about 150, and most preferably less than
about 70 Angstroms or less than about 50 angstroms.
[0014] In certain embodiments, the first electrode and/or the
second electrode have a resistivity of less than about 10.sup.-2
ohm-meters, preferably less than about 10.sup.-3 ohm-meters, more
preferably less than about 10.sup.-4 ohm-meters, and most
preferably less than about 10.sup.-5, or 10.sup.-6 ohm-meters.
Particularly preferred electrodes comprise a material such as
ruthenium, osmium, cobalt, rhodium, rubidium, lithium, sodium,
potassium, vanadium, cesium, beryllium, magnesium, calcium,
chromium, molybdenum, silicon, germanium, aluminum, iridium,
nickel, palladium, platinum, iron, copper, titanium, tungsten,
silver, gold, zinc, cadmium, indium tin oxide, carbon, or a carbon
nanotube. In certain preferred embodiments, the first electrode is
functionalized to contain a chemical group that can be derivatized
or crosslinked (e.g., a sulfate, a sulfhydryl, an amine, an
aldehyde, a carboxylic acid, a phosphate, a phosphonate, an alkene,
an alkyne, a hydroxyl group, a bromine, an iodine, a chlorine, a
light-activatable group, a group activatable by an electric
potential, etc.). The first and/or second electrode can bear a
self-assembled monolayer (SAM). Particularly preferred SAMs
comprise a compound selected from the group consisting of an
alkanethiol, a phospholipid, a bola amphiphile, and an
oligo(phenylenevinylene).
[0015] In a particularly preferred embodiment, the biological
macromolecule is attached to the first and/or to the second
electrode directly by a thiol group or through a linker bearing a
thiol group. In another particularly preferred embodiment, the
biological macromolecule is attached to the first and/or to the
second electrode directly by a phosphorothioate and/or a
phosphonate, or through a linker bearing a phosphorothioate and/or
a phosphonate. In preferred embodiments, the biological
macromolecule is attached to the first and/or to the second
electrode by a linker (e.g., DFDNB, DST, ABH, ANB-NOS, EDC,
NHS-ASA, SIA, oligo(phenylenevinylene), etc.).
[0016] The apparatus can further comprise a substrate (other than
the electrode and/or insulator) where the first electrode and the
second electrode are integrated with the substrate. In certain
embodiments, the first electrode and the second electrode are
integrated with the insulator to form a substrate. The electrodes
can be formed in essentially any desired shape (e.g. convex,
concave, textured, corrugated, patterned uniformly, randomly
patterned, etc.). Certain preferred electrode orientations include
annular, planar, and orthogonal. In certain embodiments, the first
electrode comprises a first surface and a second electrode
comprises a second surface where the first surface and the second
surface are not co-planar.
[0017] The apparatus can comprise a plurality of electrode pairs.
Thus, in certain embodiments, the first electrode and the second
electrode comprise a first electrode pair, and the molecular
sensing apparatus further comprises a second electrode pair
comprising a second first electrode and a second second electrode.
In certain embodiments, the apparatus comprises at least 3,
preferably at least 10 or 20, more preferably at least 50, 100, or
1,000, and most preferably at least 10,000 or at least 1,000,000
electrode pairs. In certain embodiments, the apparatus can comprise
electrode pairs at a density greater than about 10, preferably
greater than about 100 or 1000, more preferably greater than about
5,000, 10,000, or 50,000, and most preferably greater than about
100,000 or 1,000,000 electrode pairs per square centimeter.
[0018] In certain embodiments, the apparatus further comprises a
measurement device electrically coupled to the first electrode and
to the second electrode of at least one said electrode pair.
Preferred measurement devices measure an electromagnetic property
selected from the group consisting direct electric current,
alternating electric current, permitivity, resistivity, electron
transfer, electron tunneling, electron hopping, electron transport,
electron conductance, voltage, electrical impedance, signal loss,
dissipation factor, resistance, capacitance, inductance, magnetic
field, electrical potential, charge and magnetic potential. One
particularly preferred measurement device is a potentiostat.
[0019] The apparatus can further comprise an electrical circuit
electrically coupled to the first electrode and the second
electrode. One such circuit comprises an electrical signal gating
system (e.g. a CMOS gating system), and/or a voltage source, and/or
a multiplexor, and/or a computer.
[0020] In certain embodiments, the electrodes comprising the first
and second electrode pairs have attached the same (species of)
biological macromolecule. In certain embodiments, different
electrode pairs, have attached different biological molecules.
[0021] In certain embodiments, the first electrode and/or the
second electrode comprise a semi-conducting material. Preferred
semiconducting materials have a resistivity ranging from about
10.sup.-6 ohm-meters to about 10.sup.7 ohm-meters. Preferred
semiconducting materials include, but are not limited to silicon,
dense silicon carbide, boron carbide, Fe.sub.3O.sub.4, germanium,
silicon germanium, silicon carbide, tungsten carbide, titanium
carbide, indium phosphide, gallium nitride, gallium phosphide,
aluminum phosphide, aluminum arsenide, mercury cadmium telluride,
tellurium, selenium, ZnS, ZnO, ZnSe, CdS, ZnTe, GaSe, CdSe, CdTe,
GaAs, InP, GaSb, InAs, Te, PbS, InSb, PbTe, PbSe, and tungsten
disulfide.
[0022] In one embodiment, the apparatus comprises: a first
electrode having a first surface; a second electrode having a
second surface coplanar to the first surface; an insulator between
said first surface and said second surface; and a nucleic acid
joining the first electrode to said second electrode.
[0023] This invention also provides a method of making a molecular
sensing apparatus. In certain embodiments, the method comprises:
providing a first electrode and a second electrode separated by an
insulator; contacting the first and the second electrode with a
first solution comprising a biological macromolecule (e.g., a
nucleic acid); placing a charge on the first electrode to attract
the biological macromolecule to the first electrode where the
macromolecule attaches to the first electrode to form an attached
macromolecule; and placing a charge on the second electrode to
attract a portion of the attached macromolecule to the second
electrode to attach the macromolecule to the second electrode.
Preferred macromolecules, electrodes, electrode configurations,
insulators, measurement devices, circuits, and the like, include,
but are not limited to those described above. Where the apparatus
comprises multiple electrode pairs, the method can further comprise
contacting a second electrode pair with a second solution
comprising a second biological macromolecule; placing a charge on
the first electrode of the second electrode pair to attract the
second biological macromolecule to the first electrode of the
second electrode pair whereby the second biological macromolecule
attaches to said first electrode to form an attached second
macromolecule; and placing a charge on the second electrode of said
second electrode pair to attract a portion of said attached second
macromolecule to attach said second macromolecule to said second
electrode of said second electrode pair. The first and second
solution can be the same or different. Similarly, the first
biological macromolecule and the second biological macromolecule
can be the same or different.
[0024] In still another embodiment, this invention provides a
method of detecting an analyte. The method involves i) providing
molecular sensing apparatus comprising a first electrode and a
second electrode separated by an insulator where said first
electrode has a biological macromolecule attached thereto; ii)
contacting the attached macromolecule with said analyte whereby
said analyte binds to said macromolecule thereby forming a
macromolecule/analyte complex; iii) placing a charge on said second
electrode attract a portion of said bound analyte to said second
electrode where said second analyte is bound to the second
electrode such that the macromolecule/analyte complex forms a
connection between the first electrode and the second electrode;
and iv) detecting the connection between said first and said second
electrode. In certain embodiments, the providing comprises:
contacting the first electrode with a first solution comprising the
biological macromolecule; and placing a charge on the first
electrode whereby the charge attracts the biological macromolecule
to the electrode and the biological macromolecule attaches to the
electrode. Where multiple electrode pairs are present, the method
can involve repeating these steps for each electrode pair. The
"placing a charge" can, optionally involve placing a charge on the
first electrode opposite to the charge on the second electrode. In
certain embodiments, the "detecting" comprises detecting an
electromagnetic property selected from the group consisting of
direct electric current, alternating electric current,
permittivity, resistivity, electron transfer, electron tunneling,
electron hopping, electron transport, electron conductance,
voltage, electrical impedance, signal loss, dissipation factor,
resistance, capacitance, inductance, magnetic field, electrical
potential, charge, and magnetic potential. Preferred
macromolecules, electrodes, electrode configurations, insulators,
measurement devices, circuits, and the like, include, but are not
limited to those described above.
[0025] In still another embodiment, this invention provides a
method of detecting an analyte, where the method involves: i)
providing a molecular sensing apparatus comprising a first
electrode and a second electrode separated by an insulator where
the first electrode has a first biological macromolecule attached
thereto and the second electrode has a second biological
macromolecule attached thereto; ii) contacting the first attached
macromolecule and the second attached macromolecule with the
analyte whereby said analyte binds to the first macromolecule and
to the second macromolecule thereby forming a macromolecule/analyte
complex forming a connection between said first electrode and said
second electrode; and iii) detecting the connection between said
first and said second electrode. In certain embodiments, the
"providing" comprises contacting the first electrode with a first
solution comprising the first biological macromolecule; and placing
a charge on the first electrode whereby the charge attracts the
first biological macromolecule to the electrode and the biological
macromolecule attaches to the electrode. Similarly, in certain
embodiments, the "providing" comprises contacting the second
electrode with a solution comprising the second biological
macromolecule; and placing a charge on the second electrode whereby
the charge attracts the second biological macromolecule to the
second electrode and the second biological macromolecule attaches
to the second electrode. In certain embodiments, the "detecting"
comprises detecting an electromagnetic property selected from the
group consisting of direct electric current, alternating electric
current, permitivity, resistivity, electron transfer, electron
tunneling, electron hopping, electron transport, electron
conductance, voltage, electrical impedance, signal loss,
dissipation factor, resistance, capacitance, inductance, magnetic
field, electrical potential, charge, and magnetic potential.
Preferred macromolecules, electrodes, electrode configurations,
insulators, measurement devices, circuits, and the like, include,
but are not limited to those described above.
[0026] This invention provides still another method of detecting an
analyte. The method involves i) providing a molecular sensing
apparatus comprising a first electrode and a second electrode
separated by an insulator where a biological macromolecule forms a
connection between the first electrode and the second electrode;
ii) detecting the connection between said first and the second
electrode; iii) contacting macromolecule (binding agent) with the
analyte whereby the analyte binds to the macromolecule forming a
macromolecule/analyte complex; and iv) detecting a difference in
the connection between the first electrode and said second
electrode. In certain embodiments, the "contacting" comprises
placing a charge on the first and/or the second electrode whereby
the charge attracts the analyte to the biological macromolecule. In
certain embodiments, the "providing" comprises contacting the first
electrode with a first solution comprising the biological
macromolecule; and placing a charge on the first electrode whereby
the charge attracts the biological macromolecule to the electrode
and the biological macromolecule attaches to the electrode; and
placing a charge on the second electrode to attract a portion of
the bound macromolecule to the second electrode where the
macromolecule is bound to the second electrode such that said
macromolecule forms a connection between the first electrode and
said second electrode. In certain embodiments, the "placing charge"
comprises placing a charge on said first electrode opposite to the
charge on said second electrode. The "detecting" can comprise
detecting an electromagnetic property selected from the group
consisting of direct electric current, alternating electric
current, permitivity, resistivity, electron transfer, electron
tunneling, electron hopping, electron transport, electron
conductance, voltage, electrical impedance, signal loss,
dissipation factor, resistance, capacitance, inductance, magnetic
field, electrical potential, charge and magnetic potential. In
particularly preferred embodiments, the biological macromolecule is
attached to said first electrode by an electrically conductive
linker. In certain embodiments, the binding agent is a nucleic acid
and the analyte is a protein or a protein complex. Preferred
macromolecules, electrodes, electrode configurations, insulators,
measurement devices, circuits, and the like, include, but are not
limited to those described above.
[0027] Any of the methods and devices described herein include
embodiments where the binding agents are not joined to the first
electrode and/or the second electrodes a second or third nucleic
acid. Thus, in such embodiments, where the binding agent is a
nucleic acid, a single nucleic acid molecule spans the first and
second electrode and linkers or functional groups, if present, are
not themselves nucleic acids.
[0028] Definitions
[0029] The term "biosensor" refers to a sensor that uses a
biological macromolecule (e.g. nucleic acid, carbohydrate, protein,
antibody, etc.) to specifically recognize/bind to a target analyte.
The term "molecular sensing apparatus" is used interchangeably with
the term "biosensor".
[0030] The term "biological macromolecule" as used herein refers to
a biological molecule such as a nucleic acid, protein, antibody,
carbohydrate, polysaccharide, lipid, and the lice.
[0031] The term "electrically conductive" wherein used with
reference to a linker, molecule or molecular complex refers to the
ability of that linker, molecule or molecular complex to pass
charge through itself. Preferred electrically conductive molecules
have a resistivity lower than about 10.sup.-3 more preferably lower
than about 10.sup.-4, and most preferably lower than about
10.sup.-6 or 10.sup.-7 ohm-meters.
[0032] The term "electrically coupled" binding agent and an
electrode refers to an association between that binding agent and
the electrode such that electrons can move from the binding agent
to the electrode or from the electrode to the binding agent.
Electrical coupling can include direct covalent linkage between the
binding agent and the electrode, indirect covalent coupling (e.g.
via a linker), direct or indirect ionic bonding between the binding
agent and the electrode, or other bonding (e.g. hydrophobic
bonding). In addition, no actual bonding may be required and the
binding agent can simply be contacted with the electrode
surface.
[0033] The term "sensor element" as used herein refers to a pair of
electrodes (e.g. first electrode 10 and second electrode 12) and
associated binding agent(s) 14 that, when bound by an analyte form
a molecular complex that spans the pair of electrodes.
[0034] The terms "polypeptide", "peptide" and "protein" are used
interchangeably herein to refer to a polymer of amino acid
residues. The terms apply to amino acid polymers in which one or
more amino acid residue is an artificial chemical analogue of a
corresponding naturally occurring amino acid, as well as to
naturally occurring amino acid polymers.
[0035] The term "nucleic acid" as used herein refers to a
deoxyribonucleotide or ribonucleotide in either single- or
double-stranded form. The term encompasses nucleic acids, i.e.,
oligonucleotides, containing known analogues of natural nucleotides
which have similar or improved binding properties, for the purposes
desired, as the reference nucleic acid. The term also encompasses
nucleic-acid-like structures with synthetic backbones. DNA backbone
analogues provided by the invention include phosphodiester,
phosphorothioate, phosphorodithioate, methylphosphonate,
phosphoramidate, alkyl phosphotriester, sulfamate, 3'-thioacetal,
methylene(methylimino), 3'-N-carbamate, morpholino carbamate, and
peptide nucleic acids (PNAs); see Oligonucleotides and Analogues, a
Practical Approach, edited by F. Eckstein, IRL Press at Oxford
University Press (1991); Antisense Strategies, Annals of the New
York Academy of Sciences, Volume 600, Eds. Baserga and Denhardt
(NYAS 1992); Milligan (1993) J. Med. Chem. 36:1923-1937; Antisense
Research and Applications (1993, CRC Press). PNAs contain non-ionic
backbones, such as N-(2-aminoethyl) glycine units. Phosphorothioate
linkages are described in WO 97/03211; WO 96/39154; Mata (1997)
Toxicol. Appl. Pharmacol. 144:189-197. Other synthetic backbones
encompassed by the term include methyl-phosphonate linkages or
alternating methylphosphonate and phosphodiester linkages
(Strauss-Soukup (1997) Biochemistry 36: 8692-8698), and
benzylphosphonate linkages (Samstag (1996) Antisense Nucleic Acid
Drug Dev 6: 153-156). The term nucleic acid is used interchangeably
with gene, cDNA, mRNA, oligonucleotide primer, probe and
amplification product.
[0036] The term "antibody" refers to a polypeptide substantially
encoded by an immunoglobulin gene or immunoglobulin genes, or
fragments thereof which specifically bind and recognize an analyte
(antigen). The recognized immunoglobulin genes include the kappa,
lambda, alpha, gamma, delta, epsilon and mu constant region genes,
as well as the myriad immunoglobulin variable region genes. Light
chains are classified as either kappa or lambda. Heavy chains are
classified as gamma, mu, alpha, delta, or epsilon, which in turn
define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE,
respectively. An exemplary immunoglobulin (antibody) structural
unit comprises a tetramer. Each tetramer is composed of two
identical pairs of polypeptide chains, each pair having one "light"
(about 25 kD) and one "heavy" chain (about 50-70 kD). The
N-terminus of each chain defines a variable region of about 100 to
110 or more amino acids primarily responsible for antigen
recognition. The terms variable light chain (V.sub.L) and variable
heavy chain (V.sub.H) refer to these light and heavy chains
respectively.
[0037] Antibodies exist e.g., as intact immunoglobulins or as a
number of well-characterized fragments produced by digestion with
various peptidases. Thus, for example, pepsin digests an antibody
below the disulfide linkages in the hinge region to produce
F(ab)'.sub.2, a dimer of Fab which itself is a light chain joined
to V.sub.H--C.sub.H1 by a disulfide bond. The F(ab)'.sub.2 may be
reduced under mild conditions to break the disulfide linkage in the
hinge region, thereby converting the F(ab)'.sub.2 dimer into an
Fab' monomer. The Fab' monomer is essentially an Fab with part of
the hinge region (see, Fundamental Immunology, Third Edition, W. E.
Paul, ed., Raven Press, N.Y. 1993). While various antibody
fragments are defined in terms of the digestion of an intact
antibody, one of skill will appreciate that such fragments may be
synthesized de novo either chemically or by utilizing recombinant
DNA methodology. Thus, the term antibody, as used herein, also
includes antibody fragments either produced by the modification of
whole antibodies, those synthesized de novo using recombinant DNA
methodologies (e.g., single chain Fv), and those found in display
libraries (e.g. phage display libraries).
[0038] The phrases "hybridizing specifically to" or "specific
hybridization" or "selectively hybridize to", refer to the binding,
duplexing, or hybridizing of a nucleic acid molecule preferentially
to a particular nucleotide sequence under stringent conditions when
that sequence is present in a complex mixture (e.g., total
cellular) DNA or RNA.
[0039] The term "stringent conditions" refers to conditions under
which a probe will hybridize preferentially to its target sequence,
and to a lesser extent to, or not at all to, other sequences.
"Stringent hybridization" and "stringent hybridization wash
conditions" in the context of nucleic acid hybridization
experiments such as Southern and Northern hybridizations are
sequence dependent, and are different under different environmental
parameters. An extensive guide to the hybridization of nucleic
acids is found in Tijssen (1993) Laboratory Techniques in
Biochemistry and Molecular Biology--Hybndization with Nucleic Acid
Probes part I chapter 2 Overview of principles of hybridization and
the strategy of nucleic acid probe assays, Elsevier, New York.
Generally, highly stringent hybridization and wash conditions are
selected to be about 5.degree. C. lower than the thermal melting
point (T.sub.m) for the specific sequence at a defined ionic
strength and pH. The T.sub.m is the temperature (under defined
ionic strength and pH) at which 50% of the target sequence
hybridizes to a perfectly matched probe. Very stringent conditions
are selected to be equal to the T.sub.m for a particular probe.
[0040] An example of stringent hybridization conditions for
hybridization of complementary nucleic acids which have more than
100 complementary residues on a filter in a Southern or northern
blot is 50% formamide with 1 mg of heparin at 42.degree. C., with
the hybridization being carried out overnight. An example of highly
stringent wash conditions is 0.15 M NaCl at 72.degree. C. for about
15 minutes. An example of stringent wash conditions is a
0.2.times.SSC wash at 65.degree. C. for 15 minutes (see, Sambrook
et al. (1989) Molecular Cloning--A Laboratory Manual (2nd ed.) Vol.
1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor Press, NY,
(Sambrook et al.) supra for a description of SSC buffer). Often, a
high stringency wash is preceded by a low stringency wash to remove
background probe signal. An example of a medium stringency wash for
a duplex of, e.g., more than 100 nucleotides, is 1.times.SSC at
45.degree. C. for 15 minutes. An example of a low stringency wash
for a duplex of, e.g., more than 100 nucleotides, is 4-6.times.SSC
at 40.degree. C. for 15 minutes. In general, a signal to noise
ratio of 2.times. (or higher) than that observed for an unrelated
probe in the particular hybridization assay indicates detection of
a specific hybridization. Nucleic acids which do not hybridize to
each other under stringent conditions are still substantially
identical if the polypeptides which they encode are substantially
identical. This occurs, e.g., when a copy of a nucleic acid is
created using the maximum codon degeneracy permitted by the genetic
code.
[0041] In one particularly preferred embodiment, stringent
conditions are characterized by hybridization in 1 M NaCl, 10 mM
Tris-HCl, pH 8.0, 0.01% Triton X-100, 0.1 mg/ml fragmented herring
sperm DNA with hybridization at 45.degree. C. with rotation at 50
RPM followed by washing first in 0.9 M NaCl, 0.06 M NaH2PO4, 0.006
M EDTA, 0.01% Tween-20 at 45.degree. C. for 1 hr, followed by 0.075
M NaCl, 0.005 M NaH2PO4, 0.5 mM EDTA at 45.degree. C. for 15
minutes.
[0042] A "high resistivity plastic" refers to a plastic with a
resistivity greater than about 10.sup.-3 ohm-meters, more
preferably greater than about 10.sup.-2 ohm-meters, and most
preferably greater than about 10.sup.-1, 1, or 10 ohm-meters.
BRIEF DESCRIPTION OF THE DRAWINGS
[0043] FIGS. 1A through 1D illustrate several variations on a basic
biosensor of this invention. The sensor element comprises two
electrodes 10 and 12 connected by a binding agent (e.g. a
biomolecule). Binding of the analyte to the binding agent forms a
binding agent/analyte complex spanning the electrodes. The complex
is easily detected using, e.g. electrical means. The two electrodes
can be separated by a spacer 16 and can, optionally be fabricated
on a substrate 24 (FIG. 1D).
[0044] FIG. 2 illustrates the difference between a sandwich
configuration and one embodiment of a configuration that is not
sandwiched. In the sandwich configuration (device "A") the spacer
16 overlays all or a portion of the first electrode 10, and the
second electrode 12 overlays (or is adjacent) all or a part of the
opposite surface of the spacer 16. In device "B", the two
electrodes and spacer do not form a "sandwich configuration". While
the spacer 16 overlays all or a portion of the first electrode 10,
the second electrode 12 does not overlay all or a part of the
opposite surface of the spacer. In both embodiments, the device is
shown on a substrate 24 with a binding agent 14.
[0045] FIG. 3 illustrates two other embodiments where the two
electrodes and spacer do not form a sandwich configuration. In
device "A" the first electrode 10 and the second electrode 12 are
both in contact with the same face of the spacer 16. In device "B"
the first electrode 10 contacts the spacer 16, while the second
electrode 12 does not contact spacer 16 at all.
[0046] FIG. 4 illustrates one embodiment where the two electrodes
(10 and 12) and spacer 16 do not form a sandwich configuration. In
the embodiment illustrated, a conductor 10 is disposed on top of
spacer 16, which in turn, is disposed on top of insulator 26 that
overlays substrate 24. The second conductor 12 is disposed adjacent
to the conductor 10/spacer 16 stack and the binding agent 14
connects conductor 10 to conductor 12. Spacer/insulators 16 and 26
can be the same or different materials or can each be a combination
of materials. The device can easily be formed by depositing the
various materials (e.g. using a photolithographic process) at an
angle .theta. as illustrated.
[0047] FIGS. 5A and 5B illustrate embodiments where the two
electrodes (10 and 12) and the spacer 16 do not form a sandwich
configuration and the first electrode 10 overhangs the spacer 16.
This is readily accomplished by depositing the electrode material
at an angle .theta. as illustrated (see, e.g., FIG. 5A) and/or by
etching the spacer 16 out from underneath part of conductor 10 (see
FIG. 5B).
[0048] FIG. 6 illustrates a variety of embodiments (devices "A"
through "E") that do not have a sandwich configuration. The
spacer/insulator layer 26 is optional.
[0049] FIG. 7 illustrates a variety of embodiments (devices "F"
through "I") that do not have a sandwich configuration. The
spacer/insulator layer 26 is optional.
[0050] FIGS. 8A and 8B illustrate an embodiment of the biosensor
comprising two binding agents, 14a and 14b, one on each electrode
(FIG. 8B). The two binding agents are bound by the analyte forming
a binding agent/analyte complex spanning the electrodes. The
complex is easily detected using, e.g. electrical means.
[0051] FIGS. 9A and 9B illustrate an embodiment of the biosensor
comprising a binding agent attached to a first electrode 10 of a
pair of electrodes (FIG. 9A). The analyte binds to the binding
agent and to the second electrode 12 analyte forming a binding
agent/analyte complex spanning the electrodes (FIG. 9B). The
complex is easily detected using, e.g. electrical means.
[0052] FIGS. 10A and 10B illustrate a simple planar sensor array
according to this invention. FIG. 10A shows a top view, while FIG.
10B illustrates a side view.
[0053] FIG. 11 illustrates an aggregation of sensor arrays
according to this invention.
[0054] FIG. 12A through 12C illustrate various sensor
embodiments.
[0055] FIG. 13 illustrates two sensor elements (e.g., a subset of a
sensor array) sharing one common electrode. In the illustrated
embodiments, the first conductor 10, spacer 16, and second
conductor 12 do not form a sandwich configuration. In device B, the
spacer 16 has been partially etched out to provide an "air gap"
between the electrodes. A sensor array can readily be fabricated
containing thousands of such sensor elements.
[0056] FIG. 14 illustrates regions of a sensor array comprising two
sensor elements where each sensor element has an independent first
conductor 10 and second conductor 12. In the illustrated
embodiments, the first conductor 10, spacer 16, and second
conductor 12 do not form a sandwich configuration. In device D, the
spacer 16 has been partially etched out to provide an "air gap"
between the electrodes.
[0057] FIG. 15 illustrates a section of a sensor array of this
invention where the sensor elements are provided in a stepped
configuration.
[0058] FIG. 16 illustrates a stepped configuration for a sensor
array of this invention. In the illustrated embodiments, each
sensor element
[0059] FIG. 17 is a schematic a diagram of a support having an
array of electrode pairs (sensor elements) controlled by a
computer.
[0060] FIG. 18 is a schematic diagram of a support having an array
of electrode pairs (sensor elements).
[0061] FIG. 19 is a schematic diagram of a support having an array
of electrode pairs and computer system for controlling the
energization of each electrode pair (sensor element).
[0062] FIG. 20 is a schematic diagram of a support having an array
of electrode pairs and a computer system with a plurality of
voltage sources and multiplexers for controlling the energization
of each electrode pair (sensor element).
[0063] FIG. 21 is a diagram of a support having an array of
electrode pairs and a computer system with a plurality of switched
voltage sources for controlling the energization of each electrode
pair (sensor element).
[0064] FIG. 22 illustrates effects of deposition angle on device
configuration.
[0065] FIG. 23 illustrates device fabrication comprising a
deposition step, followed by an etching step, followed by a second
deposition.
[0066] FIGS. 24A, 24B, 24C, and 24D illustrate the deposition of
alternating conductor and insulator layers.
[0067] FIG. 25 illustrates the use of a biosensor to detect
protein/DNA interactions. A biosensor comprising a nucleic acid 14
is hybridized to a second nucleic acid 24 to form a double-stranded
nucleic acid spanning two electrodes. Binding of a protein analyte
20 (e.g. DNA binding protein) to the nucleic acid changes
conductance of the nucleic acid thereby producing a detectable
signal.
DETAILED DESCRIPTION
[0068] This invention pertains to a novel sensors (biosensors) that
are useful for detecting a wide range of analytes. The sensors
utilize a binding agent (e.g. a biomolecule) specifically bind to
one or more target analytes and thereby confer specificity and
selectivity. In preferred embodiments, the binding agent (e.g.
biomolecule) spans a gap between two electrodes. Binding of the
target analyte changes conductivity, or other electrical
properties, of the sensor thereby facilitating ready detection of
the binding event and thus detection and/or quantitation of the
bound analyte. Because the biosensors of this invention provide a
change in conductance or charge flow when bound by the target
analyte, they are easily read using electronic/electrochemical
means and do not require the use of detectable labels or external
electron donors or acceptors.
[0069] I. Sensor Element Configuration.
[0070] One embodiment of a basic biosensor (molecular sensing
apparatus) of this invention is schematically illustrated in FIG.
1. The sensor comprises a first electrode 10, a second electrode
12, and a binding agent (e.g. biomolecule) 14 spanning the gap
between the two electrodes. The two electrodes can be separated by
an air gap, however, in preferred embodiments, the electrodes are
separated by a spacer 16 (e.g. an insulator, a dielectric, or a
semiconductor). The binding agent 14 can be directly bound to the
electrodes or it can be coupled to the first electrode 10 and/or
the second electrode 12 through one or more linkers or functional
groups 18. The binding agent 14 is attached to the electrodes in a
manner that leaves sufficient area of the sensor molecule free to
bind with its "cognate" target molecule 20 (the target analyte).
When the binding agent 14 binds its cognate target molecule 20 a
binding agent/target molecule complex is formed whose conductivity
is different than the conductivity of the binding agent 14 alone.
This change in conductivity is readily detected indicating the
presence and/or concentration of the target molecule 20.
[0071] It was a surprising discovery of this invention that, in
certain instances, where the electrodes are spaced sufficiently
close together so that a biological molecule can span the two
electrodes (a molecular distance), leakage current through the
separating spacer/insulator can interfere with the conductivity
measurement of the binding agent. This problem is overcome by the
use of configurations in which the first electrode, the spacer
(e.g., insulator, dielectric, semiconductor, etc.), and the second
electrode do not form a sandwich configuration. A sandwich
configuration refers to an electrode configuration where the spacer
(e.g. insulator, diaelectric, semiconductor, etc.), overlays all or
a portion of the first electrode, and the second electrode overlays
all or a part of the opposite surface of the spacer (see, e.g.,
FIG. 2). FIG. 2 (device "B") and FIG. 3 illustrate several
electrode/spacer configurations that are not sandwich
configurations.
[0072] FIG. 4 schematically illustrates one embodiment where the
two electrodes (10 and 12) and the spacer 16 do not form a sandwich
configuration. In the embodiment illustrated, a substrate 24 is
overlayed with an insulator 26 (e.g. spacer, dielectric, etc.).
Insulater 26 is overlayed with spacer 16 which, in turn, is
overlayed with conductor 10. The second conductor 12 is disposed
adjacent to the conductor 10/spacer 16 stack and the binding agent
14 connects conductor 10 to conductor 12. Spacer/insulators 16 and
26 can be the same or different materials or can each be a
combination of materials. The device can easily be formed by
depositing the various materials (e.g. using a photolithographic
process) at an angle .theta. as illustrated.
[0073] FIGS. 5A and 5B illustrate embodiments where the two
electrodes (10 and 12) and the spacer 16 do not form a sandwich
configuration and the first electrode 10 overhangs the spacer 16.
This is readily accomplished by depositing the electrode material
at an angle .theta. as illustrated and/or by etching the spacer 16
out from underneath part of conductor 10 (see FIG. 5B.
[0074] FIGS. 6 and 7 also illustrate a variety of embodiments
(devices "A" through "I") that do not have a sandwich
configuration. These embodiments are intended to be illustrative
and not limiting. Using the teaching provided herein, numerous
other sandwich and non-sandwich configurations can be fabricated by
one of skill in the art.
[0075] In one embodiment, the binding agent 14 is a single-stranded
nucleic acid. The nucleic acid is derivatized at each terminus with
a linker that physically and electrically couples the nucleic acid
to the respective electrodes 10 and 12 such that the nucleic acid
spans the gap between the electrodes. Single-stranded nucleic acids
are essentially nonconductive. However, when the nucleic acid
binding agent is contacted with a complementary nucleic acid
analyte under conditions that permit nucleic acid hybridization,
the analyte nucleic acid binds to the sensor nucleic acid via
complementary base pairing to form a double stranded hybrid duplex
spanning the electrodes. This double stranded duplex is
electrically conductive. The change in conductivity caused by such
binding is readily detected using electrical/electrochemical
means.
[0076] The binding agent is not limited to a nucleic acid. Any
number of other binding agents can also be used in such a
biosensor. Generally, binding agents are selected that are capable
of specifically binding to a particular target analyte. Such
binding agents include, but are not limited to nucleic acids
(including, but not limited to single stranded DNA or RNA, double
stranded DNA or RNA, peptide nucleic acids, phosphorothioates, and
the like), proteins, antibodies, lectins, sugars, lipids,
polysaccharides, and the like.
[0077] While, in preferred embodiments, binding agents are utilized
that are nonconductive by themselves, but form an electrically
conductive complex when bound to the target analyte. The sensors of
this invention are not limited to such molecules. In certain
embodiments it is sufficient that the analyte/binding agent complex
simply show a different conductivity than the binding agent
alone.
[0078] Alternatively, where the analyte/binding agent complex shows
the same conductivity as the binding agent alone, it is possible to
use various chemical agents that intercalate into the
analyte/binding agent complex and change the effective conductivity
of that complex. There are typically intercalation sites, or fewer
sites afforded by the binding agent alone. Thus, the analyte
binding complex, by intercalating a greater number of such agents
shows a different conductivity.
[0079] Intercalating reagents that change the conductivity of a
biomolecule or molecular complex are well known to those of skill
in the art. Such intercalators include, but are not limited to
redox-active cations (e.g. Ru(NH.sub.3).sub.6.sup.3+ and various
transition metal/ligand complexes. Transition metals are those
whose atoms have an incomplete shell of electrons. Suitable
transition metals for use in the invention include, but are not
limited to, cadmium (Cd), magnesium (Mg), copper (Cu), cobalt (Co),
palladium (Pd), zinc (Zn), iron (Fe), ruthenium (Ru), rhodium (Rh),
osmium (Os), rhenium (Re), platinium (Pt), scandium (Sc), titanium
(Ti), Vanadium (V), chromium (Cr), manganese (Mn), nickel (Ni),
Molybdenum (Mo), technetium (Tc), tungsten (W), and iridium (Ir).
That is, the first series of transition metal, the platinum metals
(Ru, Rh, Pd, Os, Ir and Pt), along with Re, W, Mo and Tc, are
preferred. Particularly preferred are ruthenium, rhenium, osmium,
platinium and iron.
[0080] The transition metals are complexed with a variety of
ligands to form suitable transition metal complexes, as is well
known in the art. Suitable ligands include, but are not limited to,
--NH.sub.2; pyridine; pyrazine; isonicotinamide; imidazole;
bipyridine and substituted derivative of bipyridine;
phenanthrolines, particularly 1,10-phenanthroline (abbreviated
phen) and substituted derivatives of phenanthrolines such as
4,7-dimethylphenanthroline; dipyridophenazine;
1,4,5,8,9,12-hexaazatriphenylene (abbreviated hat);
9,10-phenanthrenequinone diimine; 1,4,5,8-tetraazaphenanthrene
(abbreviated tap); 1,4,8,11-tetra-azacyclotetradecane;
diaminopyridine (abbreviated damp); porphyrins and substituted
derivatives of the porphyrin family.
[0081] Such intercalating reagents can also be used to detect
mismatches between the binding agent and the target analyte. Thus,
for example where the binding agent and the analyte are nucleic
acids, intercalating reagents comprising dimeric naphthyridines
will specifically intercalate and localize where there is a G-G
mismatch between the binding reagent and the target analyte (see,
e.g., Nakatani et al. (2001) Nature/Biotechnology, 19(1): 51-55).
Such mismatch specific reagents can be used to detect or screen for
single nucleotide polymorphisms (SNPs).
[0082] While FIG. 1 illustrates essentially a single sensor element
of this invention, various embodiments contemplate the use of a
multiplicity of sensor elements. Thus, in various embodiments,
there can exist multiple binding agents 14 spanning a single pair
of electrodes and/or a multiplicity of electrode pairs 20 each
electrode pair being spanned by one or more binding agents 14.
Because of the small size of the sensor element, a large number of
sensor elements can be placed in a relatively small area (e.g. on a
chip) thereby increasing sensitivity and improving signal to noise
(S/N) ratio. In addition, assays can be performed using small
quantities of sample. A single substrate/chip can incorporate a
number of different sensor elements facilitating
detection/quantification of a number of different analytes.
[0083] The sensor elements can adopt a wide variety of
configurations. Thus, for example, in another embodiment
illustrated in FIGS. 8A and 8B, the electrodes are not spanned by a
single binding agent. Rather, a first binding agent 14a is attached
to the first electrode 10 and a second binding agent 14b is
attached to the second electrode 12 (FIG. 8A). Binding of the
analyte 20 to the two binding agents creates an electrically
conductive moiety that spans the gap between the two electrodes
allowing current to flow between the electrodes and thereby
facilitating detection/quantification of the bound analyte.
[0084] Thus, for example, in one embodiment, the first and second
binding agents are each nucleic acids complementary to half of the
target analyte. When the analyte contacts the binding agents under
conditions permitting hybridization, the two binding agents
hybridize to the analyte forming a double-stranded nucleic acid
spanning the two electrodes (see, e.g., FIG. 8B). While this is
illustrated with electrodes 10 and 12 and spacer 16 forming a
sandwich configuration, a similar approach can be taken in a
configuration that is not a sandwich configuration.
[0085] Still another preferred embodiment is illustrated in FIGS.
9A and 9B. In this embodiment, a binding agent 14 is attached to a
first electrode 10 (FIG. 9A). The target analyte is tagged with a
moiety that causes the analyte to interact with and/or bind to a
second electrode. In use, the analyte 20 binds to, e.g. the second
electrode 12 and is bound by the biological molecule 14. Together
the binding agent 14 and the analyte 20 bridge the gap between the
electrodes resulting in a detectable change in conductance. Again,
while this is illustrated with electrodes 10 and 12 and spacer 16
forming a sandwich configuration, a similar approach can be taken
in a configuration that is not a sandwich configuration.
[0086] In certain embodiments, the analyte is allowed to contact
the binding agent and form a binding agent/analyte complex. Then
application of a charge to the second electrode (and, optionally,
an opposite charge to the first electrode) draws the analyte or a
portion thereof to the second electrode whereby the analyte, or a
linker or functional group of the analyte and/or the electrode
causes the analyte to be linked to the second electrode thereby
forming the analyte/binding agent complex spanning the two
electrodes.
[0087] These configurations are simply illustrative of certain
preferred embodiments of this invention. Using the teaching
provided herein, other sensor element configurations can be readily
developed by one of ordinary skill in the art.
[0088] While each electrode (electrode pair) can bear a single
binding agent 14, typically, each electrode (electrode pair) bears
a plurality of binding agents 14. Thus, in preferred embodiments,
each electrode or electrode pair bears at least two, preferably at
least 10, more preferably at least 50, still more preferably at
least 100, and most preferably at least 1,000, at least 10,000, at
least 100,0000, or at least 1,000,000 binding agents (e.g.,
biomolecules) 14.
[0089] The electrodes comprising an electrode pair (sensor element)
can be of any convenient dimension. In preferred embodiments, the
electrodes comprising an electrode pair are spaced such that the
analyte and/or the analyte/binding agent combination span the gap
between the electrodes. In certain embodiments, the electrodes are
separated by distance ranging from about of 1 to about 10.sup.10
Angstroms, preferably from about 10 to about 10.sup.5 Angstroms,
more preferably from about 25 to about 10.sup.4 Angstroms, and most
preferably from about 40 to about 10.sup.2 angstroms. Preferred
interelectrode spacings are less than about 200 angstroms,
preferably less than about 150 angstroms, more preferably less than
about 100 angstroms, and most preferably less than about 50, about
40 or about 30 angstroms.
[0090] The gap between the electrodes can be an air gap, filled
with oxygen or with an inert gas (e.g. argon, etc.), a vacuum, or
the gap can be filled with an insulator, semiconductor, or a
dielectric. In preferred embodiments, the gap between the
electrodes is filled with an insulator. Preferred insulators
include, elements, compounds or substances that have resistivity
greater than about 10.sup.-3, preferably greater than about
10.sup.-2 ohm-meters, more preferably greater than about 10.sup.-1
ohm meters, and most preferably greater than about 10 ohm meters.
Particularly preferred insulators include, but are not limited to
SiO.sub.2, TiO.sub.2, ZrO.sub.2, porcelain, ceramic, glass, clay,
polystyrene, Teflon, plastics having a resistivity greater than
10.sup.-3 ohm-meters, and other high resistivity plastics,
insulating oxides or sulfides of the transition metals in the
periodic table of the elements, and the like.
[0091] The electrodes are conveniently formed from essentially any
conductive material. Preferred conductive materials have
resistivities of less than about 10.sup.-3 ohmmeters, preferably
less than about 10.sup.-4 ohm meters, more preferably less than
about 10.sup.-6 ohm meters, and most preferably less than about
10.sup.-7 ohm meters. In preferred embodiments, the electrodes are
formed from materials that include, but are not limited to
ruthenium, osmium, cobalt, rhodium, rubidium, lithium, sodium,
potassium, vanadium, cesium, beryllium, magnesium, calcium,
chromium, molybdenum, silicon, germanium, aluminum, iridium,
nickel, palladium, platinum, iron, copper, titanium, tungsten,
silver, gold, zinc, cadmium, indium tin oxide, carbon or carbon
nanotubes, and alloys or compounds of these materials.
[0092] II. Sensor Element Arrays.
[0093] Various embodiments of this invention can utilize a single
sensor element. However, in preferred embodiments, a plurality of
sensor elements are present, optionally forming an array of sensor
elements. As used herein, an array of sensor elements refers to a
plurality of sensor elements aggregated on a common substrate
and/or that share one or more common electrical connections.
[0094] The sensor element arrays can take essentially any
conformation that is convenient to the intended application. Thus,
in certain embodiments, the sensor element arrays can comprise
planar arrays of sensor elements (see, e.g., FIGS. 10A and 10B)
and/or aggregations of such arrays (see, e.g., FIG. 11).
[0095] The sensor element arrays are not limited to planar arrays.
Virtually any configuration can be obtained. Thus, for example,
sensor elements or arrays thereof can be placed on one or more
walls of a capillary, channel, or microchannel, on one or more
walls or floor of a sample well (e.g. in a multi-well plate such as
a microtiter plate), on one or more surfaces of a sensor probe
(e.g. an insertable or implantable sensor), and the like. In
certain embodiments, the sensor arrays can be stacked to provide
three-dimensional arrays.
[0096] Certain preferred configurations are illustrated in FIGS. 10
and 12A through 12C. Thus, for example, FIG. 10B illustrates a
flush-faced sensor array. The electrodes and insulators are
integrated into a multi-layer material presenting a flush surface.
Analyte(s) or solutions containing analytes pass across the surface
where the analytes are bound by the binding agent(s) 14. FIG. 12A
illustrates an embodiment where the electrodes protrude from the
intervening insulator and thereby form one or more channels.
[0097] The channels are useful for guiding reagents/analytes, and
the like, e.g. in various microfluidics devices. The binding
agent(s) attached to the electrodes form convenient "detector
domains" in such channels. Such devices are readily fabricated by
providing a multi-layer material, e.g. as described below, and
selectively etching insulator away from the electrodes.
[0098] Still another embodiment is illustrated in FIG. 12B. In this
embodiment, insulator/support is removed between the electrodes
thereby forming channels within the substrate having electrode
walls. Optional biasing electrodes 22 are illustrated in these
diagrams.
[0099] FIG. 12C illustrates a closed channel or well
(cross-section) in which sensor element arrays are present in two
walls of the channel.
[0100] FIG. 13 illustrates two sensor elements sharing one common
electrode. In the illustrated embodiments, the first conductor 10,
spacer 16, and second conductor 12 do not form a sandwich
configuration. In device B, the spacer 16 has been partially etched
out to provide an "air gap" between the electrodes. A sensor array
can readily be fabricated containing thousands of such sensor
elements.
[0101] FIGS. 14 and 15 illustrates a region of a sensor array
comprising two sensor elements where each sensor element has an
independent first conductor 10 and second conductor 12. In the
illustrated embodiments, the first conductor 10, spacer 16, and
second conductor 12 do not form a sandwich configuration. In device
D, the spacer 16 has been partially etched out to provide an "air
gap" between the electrodes. In device F, the second conductor 12
has either been etched away or deposited such that the conductor is
not adjacent to the spacer 16. A sensor array can readily be
fabricated containing thousands of such sensor elements by simple
photolithographic deposition techniques.
[0102] FIG. 16 illustrates a stepped configuration for a sensor
array of this invention. In the illustrated embodiments, each
sensor element (first conductor 10, binding agent 14, and second
conductor 12) comprises an independent set of conductors. The
distance between each step of "molecular dimension" (e.g. small
enough that a molecule can span from one step to the next). The
stepped configuration (a configuration that is not a sandwich
configuration as illustrated) permits a large number of sensor
elements to be distributed in on a relatively small surface. In
device "H", the spacer 16 has been etched away to provide an
"overhang" between the first and the second conductors.
[0103] These configurations are simply illustrative and not
intended to be limiting. Using the teaching provided herein,
numerous other configurations will be available to one of ordinary
skill in the art.
[0104] Preferred sensor arrays comprise at least two, preferably at
least 10, more preferably at least 100, and most preferably at
least 1, 000, 10,000 or 1,000,000 sensor elements. The sensor
elements can all bear the same biological molecules 14 or various
sensor elements can bear different biological molecules and show
specificity for different analytes. Thus, in certain embodiments, a
single sensor array can detect/quantify two or more, preferably
four or more, more preferably 10 or more, still more preferably 100
or more or 1000 or more, and most preferably 10000 more, 100,000 or
more, or even 1,000,000 or more different analytes.
[0105] The electrodes comprising the sensor elements of the
array(s) can all be separate, or they can be connected in various
combinations. Thus, for example the first electrodes 10 of all of
the sensor elements or for a subset of sensor elements can be
electrically connected to form a common electrode or "switchably
connected to form various electrical connections as desired.
Similarly, additional "biasing" electrodes 22 can be connected
together or "switchably interconnected.
[0106] Numerous methods may be used for addressing the plurality of
sensor elements comprising the sensor element arrays of this
invention. Several techniques are schematically illustrated in
FIGS. 17 through 21. Shown in those figures by way of example are
four sensor elements 101, 102, 103, 104 and appropriate
instrumentation to read them, which typically is a voltammeter
incorporating a digital computer.
[0107] In FIG. 17, each sensor element (electrode pair) pair
101-104 is individually addressed by a pair of lines connected to
the voltammeter 99. By way of example, lines 105, 106 access
electrode/counterelectrode pair 101. An appropriate voltage may be
applied and conductance/resistance measured by the voltammeter at
any given time to any one or more of the pairs of lines connected
to the various electrode pairs.
[0108] To reduce the number of connections required to address the
electrode pairs, alternatives to the direct connection scheme of
FIG. 17 are provided. For example, a row-and-column accessing
scheme is illustrated in FIG. 18 for electrically energizing some
or all of the electrodes. In this scheme, one of the electrodes
201, 202 in each column of the plurality of electrode pairs is
connected to a common electrical conductor 205 on support 200, and
each of the electrodes in each row of the plurality of electrode
pairs is connected to conductor 207, 208 on the support 200.
Conductors 205, 206 connect to connections C1, C2, respectively, at
the edge of support 200 and conductors 207, 208 connect to
connections R1, R2, respectively. Each of these connections is then
connected by a separate line to the voltammeter. As a result, in
the configuration of FIG. 18, the number of required connections
and signal lines from the voltammeter has been reduced from 8 to
4.
[0109] To enable rapid and sequential energizing/reading of each
electrode pair, a computer controlled switching device is
beneficial. The configuration of FIG. 19 shows a plurality of first
electrodes connected to a first multiplexer 310. A plurality of
second electrodes are connected to a second multiplexer 320. The
first multiplexer is also connected to a first pole of a voltage
source/voltammeter 330 that typically supplies a time varying
electrical potential for cyclic voltammetry described herein. The
second multiplexer is also connected to a second pole of the
voltage source/voltammeter. Using addressing lines A0-A3
electrically connected to each of the multiplexers and connected to
latch 340, a computer processor 350 can direct the multiplexers to
selectively connect any or all of the first electrodes to the first
pole of the voltammeter, and any or all of the second electrodes to
the second pole of the voltammeter.
[0110] As shown in FIG. 20, a plurality of voltage sources are
connected through separate sets of multiplexers to each of the
electrodes. If a first electrical potential or range of electrical
potentials is required at a particular electrode pair, the
multiplexers 410, 420 associated with the voltage source 430
providing that potential are addressed by the computer processor
350, typically through a latch 340, thereby connecting that
particular voltage source to the electrode pair in question. If a
different electrical potential or range of electrical potentials is
required for another electrode pair, the multiplexers 440, 450
associated with that different voltage source 460 are addressed by
the computer processor, thereby connecting that voltage source
through the associated multiplexers 440, 450 to the electrode
pair.
[0111] If the electrode array in this embodiment has at least a
portion of the electrode pairs independently driveable, as shown in
FIG. 18 or FIG. 19, for example, one electrode pair can be driven
by one voltage source/voltammeter while another electrode pair is
simultaneously driven with another voltage source/voltammeter.
Alternatively, the two voltage sources of FIG. 20 can be replaced
with a single voltage source/voltammeter connected to both sets of
multiplexers in parallel, allowing two electrode pairs to be driven
from the same voltage source.
[0112] Instead of a duplicate set of multiplexers for each voltage
source as shown in FIG. 20, a plurality of voltage
sources/voltammeters 520, 530 can be provided as shown in FIG. 21.
These voltage sources can be connected through a computer
controlled electrical switch 510 or switches to a single set of
multiplexers 310, 320. As shown in FIG. 21, the computer would
direct switch 510 to connect a particular voltage
source/voltammeter to the multiplexers, and would also direct the
multiplexers (by signaling their address lines A0-A3) to connect
the selected voltage source to the particular electrode pair
desired.
[0113] Alternatively, the electrical potential applied to each of
the electrode pairs in any embodiment can be varied. This is of
particular benefit when a cassette having a plurality of different
sensor elements is used. Such a cassette may require a different
range of applied electrical potential at different sensor elements.
Several different embodiments capable of varying the electrical
potential applied to each electrode are contemplated.
[0114] Advantageously, a computer controlled voltage
source/voltammeter may be used. A computer controlled voltage
source/amperometer is one that can be addressed by a computer to
select a particular electrical potential/waveform to be supplied.
Alternatively it can be programmed to sequentially apply a
particular range of electrical potentials over a predetermined
time. In such a system, address lines electrically connected to the
computer and the voltage source allow the computer to program the
voltage source to produce the particular electrical potential to be
applied to the electrode pair to be energized.
[0115] Additional methods for addressing the plurality of electrode
pairs may also be used. For example, a plurality of reference
electrodes may be placed in proximity to each of the plurality of
electrode pairs in order to sense the voltage applied thereto. In
this way, additional control of the voltage waveform may be
maintained.
[0116] While the foregoing discussion was with reference to voltage
sources/amperometers, other means of driving/reading the sensor
elements can be substituted therefor. Such means include, but are
not limited to amperometers, coulometers, and the like.
[0117] III. Sensor Molecules and Target Analytes.
[0118] A) Preferred Sensor Molecules and Target Analytes.
[0119] A wide variety of binding agents (binding reagents) 14 can
be used in the devices of this invention and the analytes that can
be detected using such binding agents are virtually limitless. The
binding agents specifically bind to at least one analyte (ligand)
of interest. The binding reagents can be selected from among any
molecules known in the art to be capable of, or putatively capable
of, specifically binding an analyte of interest.
[0120] Preferred analytes of interest include, but are not limited
to a whole cell, a subcellular particle, virus, prion, viroid,
nucleic acid, protein, antigen, lipoprotein, lipopolysaccharide,
lipid, glycoprotein, carbohydrate moiety, cellulose derivative,
antibody or fragment thereof, peptide, hormone, pharmacological
agent, cell or cellular components, organic compounds,
non-biological polymer, synthetic organic molecule, organo-metallic
compounds, or an inorganic molecule present in the sample.
[0121] The sample can be derived from, for example, a solid,
emulsion, suspension, liquid or gas. Furthermore, the sample may be
derived from, for example, body fluids or tissues, water, food,
blood, serum, plasma, urine, feces, tissue, saliva, oils, organic
solvents, earth, water, air, or food products. The sample may
comprise a reducing agent or an oxidizing agent, solubilizer,
diluent, preservative, or other suitable agents.
[0122] Suitable binding agents (biological molecules) 14 include,
but are not limited to receptors, ligands for receptors, antibodies
or binding portions thereof (e.g., Fab, (Fab)'.sub.2), proteins or
fragments thereof, nucleic acids, oligonucleotides, glycoproteins,
polysaccharides, antigens, epitopes carbohydrate moieties, enzymes,
enzyme substrates, lectins, protein A, protein G, organic
compounds, organometallic compounds, lipids, fatty acids,
lipopolysaccharides, peptides, cellular metabolites, hormones,
pharmacological agents, tranquilizers, barbiturates, alkaloids,
steroids, vitamins, amino acids, sugars, nonbiological polymers,
biotin, avidin, streptavidin, organic linking compounds such as
polymer resins, lipoproteins, cytokines, lymphokines, hormones,
synthetic polymers, organic and inorganic molecules, etc.
[0123] It will be apparent from the foregoing that the binding
agent (e.g., biological molecule) 14 and its target analyte 20 can
exist as a pair of "binding partners", e.g. a ligand and its
cognate receptor, an antibody and its epitope, etc. Thus, a
biological "binding partner" or a member of a "binding pair" refers
to a molecule or composition that specifically binds other
molecules to form a binding complex such as antibody-antigen,
lectin-carbohydrate, nucleic acid-nucleic acid, biotin-avidin,
etc.
[0124] The term "specifically binds", as used herein, when
referring to a binding agent (e.g., protein, nucleic acid;
antibody, etc.), refers to a binding reaction that is determinative
of the presence binding agent heterogeneous population of proteins
and other biologics. Thus, under designated conditions (e.g.
immunoassay conditions in the case of an antibody, or stringent
hybridization conditions in the case of a nucleic acid), the
specified ligand or antibody binds to its particular "target" (e.g.
a protein or nucleic acid) and does not bind in a significant
amount to other molecules.
[0125] The binding partner(s) used in this invention are selected
based upon the targets that are to be identified/quantified. Thus,
for example, where the target is a nucleic acid the binding partner
is preferably a nucleic acid or a nucleic acid binding protein or
protein complex (see, e.g, FIG. 25). Where the target is a protein,
the binding partner is preferably a receptor, a ligand, or an
antibody that specifically binds that protein. Where the target is
a sugar or glycoprotein, the binding partner is preferably a
lectin, and so forth.
[0126] B) Preparation of Binding Partners (Capture Agents).
[0127] Methods of synthesizing or isolating suitable binding agents
are well known to those of skill in the art as explained below.
[0128] 1) Nucleic Acids
[0129] Nucleic acids for use as binding agents 14 in this invention
can be produced or isolated according to any of a number of methods
well known to those of skill in the art. In one embodiment, the
nucleic acid can be an isolated naturally occurring nucleic acid
(e.g., genomic DNA, cDNA, mRNA, etc.). Methods of isolating
naturally occurring nucleic acids are well known to those of skill
in the art (see, e.g., Sambrook et al. (1989) Molecular Cloning--A
Laboratory Manual (2nd Ed.), Vol. 1-3, Cold Spring Harbor
Laboratory, Cold Spring Harbor, N.Y.).
[0130] In a preferred embodiment, the nucleic acid is created de
novo, e.g. through chemical synthesis, e.g., according to the solid
phase phosphoramidite triester method described by Beaucage and
Caruthers (1981), Tetrahedron Letts., 22(20): 1859-1862, e.g.,
using an automated synthesizer, as described in Needham-VanDevanter
et al. (1984) Nucleic Acids Res., 12: 6159-6168. Purification of
oligonucleotides, where necessary, is typically performed by either
native acrylamide gel electrophoresis or by anion-exchange HPLC as
described in Pearson and Regnier (1983) J. Chrom. 255: 137-149. The
sequence of the synthetic oligonucleotides can be verified using
the chemical degradation method of Maxam and Gilbert (1980) in
Grossman and Moldave (eds.) Academic Press, New York, Meth.
Enzymol. 65: 499-560.
[0131] 2) Antibodies/Antibody Fragments.
[0132] Antibodies or antibody fragments for use in sensor elements
of this invention can be produces by a number of methods well known
to those of skill in the art (see, e.g., Harlow & Lane (1988)
Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, and
Asai (1993) Methods in Cell Biology Vol. 37: Antibodies in Cell
Biology, Academic Press, Inc. N.Y.). In one approach, the
antibodies are produced by immunizing an animal (e.g. a rabbit)
with an immunogen containing the epitope it is desired to
recognize/capture. A number of immunogens may be used to produce
specifically reactive antibodies. Recombinant protein is the
preferred immunogen for the production of monoclonal or polyclonal
antibodies. Naturally occurring protein may also be used either in
pure or impure form. Synthetic peptides made as well using standard
peptide synthesis chemistry (see, e.g., Barany and Merrifield,
Solid-Phase Peptide Synthesis; pp. 3-284 in The Peptides: Analysis,
Synthesis, Biology. Vol. 2: Special Methods in Peptide Synthesis,
Part A., Merrifield et al. (1963) J. Am. Chem. Soc., 85: 2149-2156,
and Stewart et al. (1984) Solid Phase Peptide Synthesis, 2nd ed.
Pierce Chem. Co., Rockford, Ill.)
[0133] Methods of production of polyclonal antibodies are known to
those of skill in the art. In brief, an immunogen is mixed with an
adjuvant and animals are immunized. The animal's immune response to
the immunogen preparation is monitored by taking test bleeds and
determining the titer of reactivity to the immunogen. When
appropriately high titers of antibody to the immunogen are
obtained, blood is collected from the animal and antisera are
prepared. Further fractionation of the antisera to enrich for
antibodies reactive to the immunogen can be done if desired. (See
Harlow and Lane, supra).
[0134] Monoclonal antibodies may be obtained by various techniques
familiar to those skilled in the art. Briefly, spleen cells from an
animal immunized with a desired antigen are immortalized, commonly
by fusion with a myeloma cell (See, Kohler and Milstein (1976) Eur.
J. Immunol. 6: 511-519). Alternative methods of immortalization
include transformation with Epstein Barr Virus, oncogenes, or
retroviruses, or other methods well known in the art. Colonies
arising from single immortalized cells are screened for production
of antibodies of the desired specificity and affinity for the
antigen, and yield of the monoclonal antibodies produced by such
cells may be enhanced by various techniques, including injection
into the peritoneal cavity of a vertebrate host. Alternatively, one
may isolate DNA sequences which encode a monoclonal antibody or a
binding fragment thereof by screening a DNA library from human B
cells according to the general protocol outlined by Huse et al.
(1989) Science, 246:1275-1281.
[0135] Antibodies fragments, e.g. single chain antibodies (scFv or
others), can also be produced/selected using phage display
technology. The ability to express antibody fragments on the
surface of viruses that infect bacteria (bacteriophage or phage)
makes it possible to isolate a single binding antibody fragment
from a library of greater than 10.sup.10 nonbinding clones. To
express antibody fragments on the surface of phage (phage display),
an antibody fragment gene is inserted into the gene encoding a
phage surface protein (pIII) and the antibody fragment-pIII fusion
protein is displayed on the phage surface (McCafferty et al. (1990)
Nature, 348: 552-554; Hoogenboom et al. (1991) Nucleic Acids Res.
19: 4133-4137).
[0136] Since the antibody fragments on the surface of the phage are
functional, phage bearing antigen binding antibody fragments can be
separated from non-binding phage by antigen affinity chromatography
(McCafferty et al. (1990) Nature, 348: 552-554). Depending on the
affinity of the antibody fragment, enrichment factors of 20
fold-1,000,000 fold are obtained for a single round of affinity
selection. By infecting bacteria with the eluted phage, however,
more phage can be grown and subjected to another round of
selection. In this way, an enrichment of 1000 fold in one round can
become 1,000,000 fold in two rounds of selection (McCafferty et al.
(1990) Nature, 348: 552-554). Thus even when enrichments are low
(Marks et al. (1991) J. Mol. Biol. 222: 581-597), multiple rounds
of affinity selection can lead to the isolation of rare phage.
Since selection of the phage antibody library on antigen results in
enrichment, the majority of clones bind antigen after as few as
three to four rounds of selection. Thus only a relatively small
number of clones (several hundred) need to be analyzed for binding
to antigen.
[0137] Human antibodies can be produced without prior immunization
by displaying very large and diverse V-gene repertoires on phage
(Marks et al. (1991) J. Mol. Biol. 222: 581-597). In one embodiment
natural V.sub.H and V.sub.L repertoires present in human peripheral
blood lymphocytes are were isolated from unimmunized donors by PCR.
The V-gene repertoires were spliced together at random using PCR to
create a scPv gene repertoire which is was cloned into a phage
vector to create a library of 30 million phage antibodies (Id.).
From this single "naive" phage antibody library, binding antibody
fragments have been isolated against more than 17 different
antigens, including haptens, polysaccharides and proteins (Marks et
al. (1991) J. Mol. Biol. 222: 581-597; Marks et al. (1993).
Bio/Technology. 10: 779-783; Griffiths et al. (1993) EMBO J. 12:
725-734; Clackson et al. (1991) Nature. 352: 624-628). Antibodies
have been produced against self proteins, including human
thyroglobulin, immunoglobulin, tumor necrosis factor and CEA
(Griffiths et al. (1993) EMBO J. 12: 725-734). It is also possible
to isolate antibodies against cell surface antigens by selecting
directly on intact cells. The antibody fragments are highly
specific for the antigen used for selection and have affinities in
the 1:M to 100 nM range (Marks et al. (1991) J. Mol. Biol. 222:
581-597; Griffiths et al. (1993) EMBO J. 12: 725-734). Larger phage
antibody libraries result in the isolation of more antibodies of
higher binding affinity to a greater proportion of antigens.
[0138] 3) Binding Proteins.
[0139] In one embodiment, the binding partner (capture agent) can
be a binding protein. Suitable binding proteins include, but are
not limited to receptors (e.g. cell surface receptors), receptor
ligands, cytokines, transcription factors and other nucleic acid
binding proteins, growth factors, etc.
[0140] The protein can be isolated from natural sources,
mutagenized from isolated proteins or synthesized de novo. Means of
isolating naturally occurring proteins are well known to those of
skill in the art. Such methods include but are not limited to well
known protein purification methods including ammonium sulfate
precipitation, affinity columns, column chromatography, gel
electrophoresis and the like (see, generally, R. Scopes, (1982)
Protein Purification, Springer-Verlag, N.Y.; Deutscher (1990)
Methods in Enzymology Vol. 182: Guide to Protein Purification,
Academic Press, Inc. N.Y.).
[0141] Where the protein binds a target reversibly, affinity
columns bearing the target can be used to affinity purify the
protein. Alternatively the protein can be recombinantly expressed
with a HIS-Tag and purified using Ni.sup.2+/NTA chromatography.
[0142] In another embodiment, the protein can be-chemically
synthesized using standard chemical peptide synthesis techniques.
Where the desired subsequences are relatively short the molecule
may be synthesized as a single contiguous polypeptide. Where larger
molecules are desired, subsequences can be synthesized separately
(in one or more units) and then fused by condensation of the amino
terminus of one molecule with the carboxyl terminus of the other
molecule thereby forming a peptide bond. This is typically
accomplished using the same chemistry (e.g., Fmoc, Tboc) used to
couple single amino acids in commercial peptide synthesizers.
[0143] Solid phase synthesis in which the C-terminal amino acid of
the sequence is attached to an insoluble support followed by
sequential addition of the remaining amino acids in the sequence is
the preferred method for the chemical synthesis of the polypeptides
of this invention. Techniques for solid phase synthesis are
described by Barany and Merrifield (1962) Solid-Phase Peptide
Synthesis; pp. 3-284 in The Peptides: Analysis, Synthesis, Biology.
Vol. 2: Special Methods in Peptide Synthesis, Part A., Merrifield
et al. (1963) J. Am. Chem. Soc., 85: 2149-2156, and Stewart et al.
(1984) Solid Phase Peptide Synthesis, 2nd ed. Pierce Chem. Co.,
Rockford, Ill.
[0144] In a preferred embodiment, the protein can also be
synthesized using recombinant DNA methodology. Generally this
involves creating a DNA sequence that encodes the binding protein,
placing the DNA in an expression cassette under the control of a
particular promoter, expressing the protein in a host, isolating
the expressed protein and, if required, renaturing the protein.
[0145] DNA encoding binding proteins or subsequences of this
invention can be prepared by any suitable method as described
above, including, for example, cloning and restriction of
appropriate sequences or direct chemical synthesis by methods such
as the phosphotriester method of Narang et al. (1979) Meth.
Enzymol. 68: 90-99; the phosphodiester method of Brown et al.
(1979) Meth. Enzymol. 68: 109-151; the diethylphosphoramidite
method of Beaucage et al. (1981) Tetra. Lett., 22: 1859-1862; and
the solid support method of U.S. Pat. No. 4,458,066.
[0146] The nucleic acid sequences encoding the desired binding
protein(s) may be expressed in a variety of host cells, including
E. coli, other bacterial hosts, yeast, and various higher
eukaryotic cells such as the COS, CHO and HeLa cells lines and
myeloma cell lines. The recombinant protein gene will be operably
linked to appropriate expression control sequences for each host.
For E. coli this includes a promoter such as the T7, trp, or lambda
promoters, a ribosome binding site and preferably a transcription
termination signal. For eukaryotic cells, the control sequences
will include a promoter and preferably an enhancer derived from
immunoglobulin genes, SV40, cytomegalovirus, etc., and a
polyadenylation sequence, and may include splice donor and acceptor
sequences.
[0147] The plasmids can be transferred into the chosen host cell by
well-known methods such as calcium chloride transformation for E.
coli and calcium phosphate treatment or electroporation for
mammalian cells. Cells transformed by the plasmids can be selected
by resistance to antibiotics conferred by genes contained on the
plasmids, such as the amp, gpt, neo and hyg genes.
[0148] Once expressed, the recombinant binding proteins can be
purified according to standard procedures of the art as described
above.
[0149] 4) Sugars and Carbohydrates.
[0150] Other binding agents suitable for sensor elements of this
invention include, but are not limited to, sugars and
carbohydrates. Sugars and carbohydrates can be isolated from
natural sources, enzymatically synthesized or chemically
synthesized. A route to production of specific oligosaccharide
structures is through the use of the enzymes which make them in
vivo; the glycosyltransferases. Such enzymes can be used as regio-
and stereoselective catalysts for the in vitro synthesis of
oligosaccharides (Ichikawa et al. (1992) Anal. Biochem. 202:
215-238). Sialyltransferase can be used in combination with
additional glycosyltransferases. For example, one can use a
combination of sialyltransferase and galactosyltransferases. A
number of methods of using glycosyltransferases to synthesize
desired oligosaccharide structures are known. Exemplary methods are
described, for instance, WO 96/3249.1, Ito et al. (1993) Pure Appl.
Chem. 65:753, and U.S. Pat. Nos. 5,352,670, 5,374,541, and
5,545,553. The enzymes and substrates can be combined in an initial
reaction mixture, or alternatively, the enzymes and reagents for a
second glycosyltransferase cycle can be added to the reaction
medium once the first glycosyltransferase cycle has neared
completion. By conducting two glycosyltransferase cycles in
sequence in a single vessel, overall yields are improved over
procedures in which an intermediate species is isolated.
[0151] Methods of chemical synthesis are described by Zhang et al.
(1999) J. Am. Chem. Soc., 121(4): 734-753. Briefly, in this
approach, a set of sugar-based building blocks is created with each
block preloaded with different protecting groups. The building
blocks are ranked by reactivity of each protecting group. A
computer program then determines exactly which building blocks must
be added to the reaction so that the sequences of reactions from
fastest to slowest produces the desired compound.
[0152] IV. Assembling a Sensor.
[0153] The biosensors of this invention can be assembled using
methods well known to those of skill in the art. In general two or
more electrodes are provided having an inter-electrode spacing
sufficiently small that the biomolecule/target analyte complex is
capable of carrying charge from one electrode to the other. The
electrode(s) are then contacted with the biomolecule(s) 14 in a
manner that facilitates the electrical coupling and physical
attachment of the biomolecule(s) to one or both electrodes
(depending on device configuration). The electrode(s) and/or the
biomolecules can be derivatized so that the molecules self
assemble/attach to the electrode.
[0154] A) Providing Two or More Electrodes.
[0155] Methods of providing electrodes closely positioned with
respect to each other are well known to those of skill in the art.
Thus, for example, electrodes can be precisely positioned using
micromanipulators, atomic force microscope (AFM) or STM tips, and
the like. In preferred embodiments, the plurality of electrodes
(optional counter electrodes) and the like are typically placed in
registered proximity to one another by mechanical means, e.g., by
using guide posts, alignment pins, guide edges, and the like. Other
systems using electrical or magnetic registration means are also
available.
[0156] In particularly preferred embodiments, the electrodes are
fabricated/positioned using micromachining processes (e.g.
photolithography) well known in the solid state electronics
industry. Commonly, microdevices are constructed from semiconductor
material substrates such as crystalline silicon, widely available
in the form of a semiconductor wafer used to produce integrated
circuits, or from glass. Because of the commonality of material(s),
fabrication of microdevices from a semiconductor wafer substrate
can take advantage of the extensive experience in both surface and
bulk etching techniques developed by the semiconductor processing
industry for integrated circuit (IC) production.
[0157] Surface etching, used in IC production for defining thin
surface patterns in a semiconductor wafer, can be modified to allow
for sacrificial undercut etching of thin layers of semiconductor
materials to create spaces or gaps. Bulk etching, typically used in
IC production when deep trenches are formed in a wafer using
anisotropic etch processes, can be used to precisely machine edges
or trenches in microdevices. Both surface and bulk etching of
wafers can proceed with "wet processing", using chemicals such as
potassium hydroxide in solution to remove non-masked material from
a wafer. For microdevice construction, it is even possible to
employ anisotropic wet processing techniques that rely on
differential crystallographic orientations of materials, or the use
of electrochemical etch stops, to define various channel
elements.
[0158] Another etch processing technique that allows great
microdevice design freedom is commonly known as "dry etch
processing". This processing technique is particularly suitable for
anistropic etching of fine structures. Dry etch processing
encompasses many gas or plasma phase etching techniques ranging
from highly anisotropic sputtering processes that bombard a wafer
with high energy atoms or ions to displace wafer atoms into vapor
phase (e.g. ion beam milling), to somewhat isotropic low energy
plasma techniques that direct a plasma stream containing chemically
reactive ions against a wafer to induce formation of volatile
reaction products.
[0159] Intermediate between high energy sputtering techniques and
low energy plasma techniques is a particularly useful dry etch
process known as reactive ion etching. Reactive ion etching
involves directing an ion containing plasma stream against a
semiconductor, or other, wafer for simultaneous sputtering and
plasma etching. Reactive ion etching retains some of the advantages
of anisotropy associated with sputtering, while still providing
reactive plasma ions for formation of vapor phase reaction products
in response to contacting the reactive plasma ions with the wafer.
In practice, the rate of wafer material removal is greatly enhanced
relative to either sputtering techniques or low energy plasma
techniques taken alone. Reactive ion etching therefore has the
potential to be a superior etching process for construction of
microdevices, with relatively high anistropic etching rates being
sustainable. The micromachining techniques described above, as well
as many others, are well known to those of skill in the art (see,
e.g., Choudhury (1997) The Handbook of Microlithography,
Micromachining, and Microfabrication), Soc. Photo-Optical Instru.
Engineer, Bard & Faulkner (1997) Fundamentals of
Microfabrication). In addition, examples of the use of
micromachining techniques on silicon or borosilicate glass chips
can be found in U.S. Pat. Nos. 5,194,133, 5,132,012, 4,908,112, and
4,891,120.
[0160] In certain embodiments, the electrodes, particularly
electrode arrays of this invention are formed as multilayer
materials, e.g. alternating layers of dieletric and conductor. When
etched, cut, or otherwise fractured, the edge of such multilayer
materials affords electrodes separated by dielectric/insulator at
extremely high density (close spacing).
[0161] Multilayer materials are widely known in the materials
community for scientific study and physics applications and their
use has been demonstrated widely (see, e.g., U.S. Pat. Nos.
4,673,623, 4,870,648, 4,915,463 and the like).
[0162] Such electrode arrays are readily fabricated using
sputtering techniques (see, e.g. U.S. Pat. Nos. 5,203,977,
5,486,277, RE37,032, 5,742,471, and the like). Sputtering is a
vacuum coating process where an electrically isolated cathode is
mounted in a chamber that can be evacuated and partially filled
with an inert gas. If the cathode material is an electrical
conductor, a direct-current high-voltage power supply is used to
apply the high voltage potential. If the cathode is an electrical
insulator, the polarity of the electrodes is reversed at very high
frequencies to prevent the formation of a positive charge on the
cathode that would stop the ion bombardment process. Since the
electrode polarity is reversed at a radio frequency, this process
is referred to as RF-sputtering.
[0163] Magnetron sputtering is a more effective form than diode
sputtering that uses a magnetic field to trap electrons in a region
near the target surface creating a higher probability of ionizing a
gas atom. The high density of ions created near the target surface
causes material to be removed many times faster than in diode
sputtering. The magnetron effect is created by an array of
permanent magnets included within the cathode assembly that produce
a magnetic field normal to the electric field. While other
sputtering techniques may be used, in particularly preferred
embodiments, magnetron sputtering, e.g. as described in U.S. Pat.
No. 5,486,277, s used to provide the electrode arrays of this
invention.
[0164] FIG. 22 illustrates how deposition angle can be used to
control the location of various conductors (electrodes). With a
deposition beam oriented normal to the substrate (conductor set
"A"), the insulator and conductors 10 and 12 can be deposited
adjacent to each other as illustrated. Use of an appropriate mask
during spacer 16 deposition the area covered by the spacer to be
precisely delineated. Then deposition of a conductive material
produces conductors 10 and 12.
[0165] The deposition angle can be varied to produce alternate
patterns. As shown in FIG. 22, conductor set "B", deposition of the
conductor material at an angle oblique to the substrate can produce
a space between conductor 12 and spacer 16.
[0166] As indicated above, the deposition techniques can be
combined with etching methods. Conductor set "C" illustrates the
result of selectively etching the spacer of conductor set "A". This
produces a "cutback" of the spacer with an air gap between
conductor 10 and conductor 12.
[0167] FIG. 23 illustrates another combination of deposition and
etching. In this instance, the substrate is uniformly coated first
with the spacer material 16 and then with the conductor material 10
(see, conductor set "A"). The conductor material 10 and spacer
material 16 are then selectively etched in one region to produce a
well or channel (see, conductor set "B"). A second deposition step
with an appropriate mask deposits conductor material 12 in the well
or channel (see, conductor set "C"). These variations are meant to
be illustrative and not limiting. Using the teaching provided
herein, one of skill will readily be able to fabricate the devices
of this invention.
[0168] B) Attachment of Biomolecules to Electrodes.
[0169] The binding agents (e.g. biomolecules) are attached to the
electrodes using methods well known to those of skill in the art.
Typically the electrode(s) and/or the binding agent(s) are
derivatized (functionalized) with reactive moieties (e.g. linkers)
that facilitate attachment of the electrode to the binding agent.
Thus, for example in certain embodiments, the binding agent bears a
reactive linker (e.g. an aliphatic thiol linker) that reacts with
the electrode surface or with a functional group attached thereto,
and/or the electrode is derivatized with a linker that binds to the
biomolecule.
[0170] The linker can be electrically conductive or it can be short
enough that electrons can pass directly or indirectly between the
electrode and the biological molecule 14.
[0171] The manner of linking a wide variety of compounds to various
surfaces is well known and is amply illustrated in the literature.
Means of coupling the biological molecules 14 will be recognized by
those of skill in the art. The linkage can be covalent, or by ionic
or other non-covalent interactions. The surface and/or the
molecule(s) may be specifically derivatized to provide convenient
linking groups (e.g. sulfur, hydroxyl, amino, etc.).
[0172] The linker(s) can be provided as a part of a derivatized
binding agent or they can be provided separately. Linkers, when not
joined to the molecules to be linked are often either hetero- or
homo-bifunctional molecules that contain two or more reactive sites
that may each form a covalent bond with the respective binding
partner (i.e. electrode surface or biological molecule). When
provided as a component the biological molecule, or attached to the
electrode, the linkers are preferably spacers having one or more
reactive sites suitable for bonding to the respective surface or
molecule.
[0173] Linkers suitable for joining molecules are well known to
those of skill in the art and include, but are not limited to any
of a variety of, a straight or branched chain carbon linker, or a
heterocyclic carbon linker, amino acid or peptide linkers, and the
like. Particularly preferred linkers include, but are not limited
to 4,4'-diphenylethyne, 4,4'-diphenylbutadiyne, 4,4'-biphenyl,
1,4-phenylene, 4,4'-stilbene, 1,4-bicyclooctane, 4,4'-azobenzene,
4,4'-benzylideneaniline, and 4,4"-telphenyl, oligophenylene
vinylene, and the like (see, e.g., U.S. Pat. No. 6,208,553).
[0174] A wide variety of such linkers comprising surface binding
groups are know to those of skill in the art and are often used to
produce self-assembling monolayers. Such groups include, but are
not limited to thiols (e.g. alkanethiols) (which bind gold and
other metals), alkyltrichlorosilane (e.g., which bind
silicon/silicon dioxide), alkane carboxylic acids (e.g., which bind
aluminum oxides), derivatives of ethylene glycol, as well as
combinations thereof (see, e.g., Perguson et al. (1993)
Macromolecules 26(22): 5870-5875; Prime et al. (1991) Science
252:1164-1167; Bain et al. (1989) Angew. Chem. 101: 522-528; Kumar
et al. (1994) Lan gmuir 10: 1498-1511; Laibinis et al. (1989)
Science 245: 845-847; Pale-Grosdemange et al. (1991) J. Am. Chem.
Soc., 113: 12-20, and the like). In particularly preferred
embodiments, the biological molecules 14 are attached to metal
electrodes using thiol linkers (e.g., alkanethiol linkers).
[0175] In certain embodiments, the binding agents are
functionalized with a chemical group, or a linker bearing a
chemical group, that can be activated by the application of an
electrical potential. Such groups are well known to those of skill
in the art and include, but are not limited to S-benzyloxycarbonyl
derivatives, S-benzyl thioethers, S-phenyl thioethers, S-4-picolyl
thioethers, S-2,2,2-trichloroethoxycarbonyl derivatives,
S-triphenylmethyl thioethers, and the like. In certain embodiments,
the binding agents are functionalized with a chemical group, or a
linker bearing a chemical group that can be activated by light of
wavelength ranging from 190 nm to 700 nm. Such chemical groups
include, but are not limited to an aryl azide, a flourinated aryl
azide, a benzophenone, and
(R,S)-1-(3,4-(methylene-dioxy)-6-nitrophenyl) ethyl
cholorformate--(MeNPOC), N-((2-pyridyl, ethyl)-4-azido)
salicylamide
[0176] In a particularly preferred embodiment the derivatized
biological molecule, in solution, is contacted with the
electrode(s). A charge is placed on the first electrode 10 to
attract the biological molecule thereto. Upon contact with the
electrode, the derivatized biological molecule binds to the
electrode. The derivatized biological molecule can bear two
linkers, one for attachment to the first electrode and one
derivatized for attachment to the second electrode. In such
embodiments, the second linker can be blocked to prevent reaction
with the first electrode. After the biological molecule has been
bound to the first molecule the linker is deprotected permitting
binding to the second electrode.
[0177] Thus, for example to span two electrodes with a biological
molecule that is a nucleic acid, the nucleic acid is derivatized
with two linkers one protected (blocked) thiol and one deprotected
(unblocked) thiol. The first electrode 12 is biased positive to
attract the nucleic acid thereto whereby the thiol linker binds to
the first electrode. The first electrode 10 is then biased negative
and the second electrode 12 is biased positive to attract the free
end of the nucleic acid to second electrode. The blocked thiol
linker is deprotected leaving that linker free to interact with the
second. This results in a nucleic acid spanning gap between the
first and the second electrode.
[0178] This assembly approach thus uses the device itself, to
direct the localization and ultimate attachment of the binding
agent. Thus, the devices of this invention are able to
electronically self-address each sensor element with a specific
binding agent. The device self-assembles itself in the sense that
no outside process, mechanism, or equipment is needed to physically
direct, position, or place a specific binding agent at a specific
location/sensor element/electrode. This self-addressing process is
both rapid and specific, and can be carried out in either a serial
or parallel manner.
[0179] The device can be serially addressed with specific binding
agent by maintaining selected sensor element(s)/electrode(s) in a
DC mode and at the opposite charge (potential) to that of a
specific binding entity. Other sensor elements/electrodes are
maintained at the same charge as the specific binding agent. In
cases where the binding agent is not in excess of the attachment
sites on electrode(s), it is necessary to activate only one other
micro-electrode to affect the electrophoretic transport to the
specific microlocation. The specific binding agent is rapidly
transported (in a few seconds, or preferably less than a second)
through the solution, and concentrated directly at the specific
electrode where can covalently bonded to the electrode surface.
[0180] The parallel process for addressing sensor
elements/electrodes simply involves simultaneously activating a
large number (particular group or line) of electrodes so that the
same specific binding entity is transported, concentrated, and
reacted with more than one specific electrode.
[0181] This approach is simply illustrative. Numerous other
approached can be used to attach the biological molecule to the
respective electrode(s). Such approaches include, but are not
limited to attachment of chemical groups to the surface through the
use of photoactivatable chemistries (see, e.g., Sundberg et al.
(1995) J. Am. Chem. Soc. 117(49):12050-12057), micro-stamping
techniques (see, e.g., Kumar et al. (1994) Langmuir
10(5):1498-1511; Kumar et al. (1993) Appl. Phys. Lett.
63(14):2002-2004), and the like.
[0182] V. Reading the Sensor.
[0183] The sensors of this invention are read using standard
methods well known to those of skill in the art. In particular, the
sensors of this invention provide a signal that is a change in
conductivity (resistivity) of the sensor element(s) as target
analytes are bound.
[0184] In preferred embodiments, the sensors of this invention are
read using techniques including, but not limited to amperommetry,
voltammetry, capacitance, and impedence. Suitable techniques
include, but are not limited to, electrogravimetry; coulometry
(including controlled potential coulometry and constant current
coulometry); voltametry (cyclic voltametry, pulse voltametry
(normal pulse voltametry, square wave voltametry, differential
pulse voltametry, Osteryoung square wave voltametry, and
coulostatic pulse techniques); stripping analysis (aniodic
stripping analysis, cathiodic stripping analysis, square wave
stripping voltammetry); conductance measurements (electrolytic
conductance, direct analysis); time-dependent electrochemical
analyses (chronoamperometry, chronopotentiometry, cyclic
chronopotentiometry and amperometry, AC polography,
chronogalvametry, and chronocoulometry); AC impedance measurement;
capacitance measurement; and photoelectrochemistry.
[0185] In a preferred embodiment, monitoring electron transfer
through the binding agent/target analyte complex is via
amperometric detection. In certain embodiments, a preferred
amperometric detector resembles the numerous enzyme-based
biosensors currently used to monitor blood glucose, for example.
This method of detection involves applying a potential (as compared
to a separate reference electrode) between the two electrodes
comprising a sensor element of this invention. Electron transfer of
differing efficiencies is induced in samples in the presence or
absence of target nucleic add; that is, where the binding agent is
a nucleic acid, the single stranded binding agent exhibits a
different rate than the probe hybridized to the target sequence.
The differing efficiencies of electron transfer result in differing
currents being generated in the electrode.
[0186] In preferred embodiments, devices for measuring electron
transfer amperometrically involves sensitive (nanoamp to picoamp)
current detection and include a means of controlling the voltage
potential, usually a potentiostat.
[0187] In other preferred embodiments, alternative electron
detection modes are utilized. For example, potentiometric (or
voltammetric) measurements involve non-faradaic (no net current
flow) processes and are utilized traditionally in pH and other ion
detectors. Similar sensors can be used to monitor electron transfer
the binding agent/target analyte complex. In addition, other
properties of insulators (such as resistance) and of conductors
(such as conductivity, impedance and capacitance) can be used to
monitor electron transfer through the binding agent/target analyte
complex. Finally, any system that generates a current (such as
electron transfer) also generates a small magnetic field, which can
be monitored in some embodiments.
[0188] In preferred embodiments, the relatively fast rates of
electron transfer through the binding agent/target analyte complex
can facilitate analysis in the frequency (time) domain and thereby
dramatically improve signal to noise (S/N) ratios. Thus, in certain
embodiments, electron transfer is initiated and detected using
alternating current (AC) methods. In general, the use of AC
techniques can result in good signals and low background noise.
Without being bound by theory, there are a number of possible
contributors to background noise, or "parasitic" signals, i.e.
detectable signals that are inherent to the system but are not the
result of the presence of the target sequence.
[0189] However, all of the contributors to parasitic noise
generally give relatively fast signals; that is, the rate of
electron transfer through the binding agent/target analyte complex
is generally significantly slower than the rate of electron
transfer of the parasitic components, such as the contribution of
charge carriers in solution, and other "short circuiting"
mechanisms. As a result, the parasitic components are generally all
phase related; that is, they exhibit a constant phase delay or
phase shift that will scale directly with frequency. The binding
agent/target analyte complex, in contrast, exhibits a time delay
between the input and output signals, which is independent of
frequency. Thus, signal produced by analyte binding will remain
relatively constant and relatively large as compared to parasitic
background. As a consequence, at different frequencies, the phase
of the system will change. This is very similar to the time domain
detection used in fluorescent systems.
[0190] This difference can be exploited in various methods to
decrease the signal to noise ratio. Accordingly, the preferred
detection methods comprise applying an AC input signal to a binding
agent/target analyte complex. The presence of the binding
agent/target analyte complex is detected via an output signal
characteristic of electron transfer through the binding
agent/target analyte complex; that is, the output signal is
characteristic of the binding agent/target analyte complex rather
than the parasitic components or unbound binding agent. Thus, for
example, the output signal will exhibit a time delay dependent on
the rate of electron transfer through the binding agent/target
analyte complex.
[0191] In certain preferred embodiments, the input signals are
applied at a plurality of frequencies, since this again allows the
distinction between true signal and noise. "Plurality" in this
context means at least two, and preferably more, frequencies. In
general, the AC frequencies will range from about 0.1 Hz to about
10 mHz, with from about 1 Hz to 100 KHz being preferred.
[0192] In certain preferred embodiments, data analysis is preformed
in the time domain (frequency domain). Thus, for example, cyclic
voltammetry is performed where the signal is analyzed at a harmonic
of the fundamental frequency. Such measurements can significantly
improve the signal to noise (S/N) ratio.
[0193] In preferred embodiments, a cyclic (e.g., sinusoidal
sweeping voltage) is applied to the electrode. The response of the
binding agent/target analyte complex to the sinusoidal voltage is
selectively detected at a harmonic of the fundamental frequency of
the cyclic voltage rather than at the fundamental frequency. As a
result, a complete frequency spectrum can be obtained within one
cycle.
[0194] The step of selectively detecting the voltammetric response
comprises the step of selectively detecting a current flowing
through the binding agent/target analyte complex at a harmonic of
the fundamental frequency. Preferably the harmonic comprises at
least one harmonic of the current above the fundamental frequency.
Typically, the signal is monitored at harmonics at and above the
second harmonic of the fundamental frequency. In general, the step
of selectively detecting the voltammetric response comprises the
step of detecting a plurality of higher harmonics of the
fundamental frequency within a frequency spectrum of a current
flowing through the analyte, either through the use of multiple
lock-in detectors, or via data acquisition in the time domain,
followed by, e.g., Fourier transformation and convolution via
computer based methods. Methods of cyclic voltammetry are known to
those of skill in the art and describe in detail in U.S. Pat. Nos.
6,208,553 and 5,958,215
[0195] VI. Analyte Detection/Quantification.
[0196] A) Sample Preparation.
[0197] Virtually any sample can be analyzed using the devices and
methods of this invention. Such samples include, but are not
limited to body fluids or tissues, water, food, blood, serum,
plasma, urine, feces, tissue, saliva, oils, organic solvents,
earth, water, air, or food products. In a preferred embodiment, the
sample is a biological sample. The term "biological sample", as
used herein, refers to a sample obtained from an organism or from
components (e.g., cells) of an organism. The sample may be of any
biological tissue or fluid. Frequently the sample will be a
"clinical sample" which is a sample derived from a patient. Such
samples include, but are not limited to, sputum, cerebrospinal
fluid, blood, blood fractions (e.g. serum, plasma), blood cells
(e.g., white cells), tissue or fine needle biopsy samples, urine,
peritoneal fluid, and pleural fluid, or cells therefrom. Biological
samples may also include sections of tissues such as frozen
sections taken for histological purposes.
[0198] Biological samples, (e.g. serum) may be analyzed directly or
they may be subject to some preparation prior to use in the assays
of this invention. Such preparation can include, but is not limited
to, suspension/dilution of the sample in water or an appropriate
buffer or removal of cellular debris, e.g. by centrifugation, or
selection of particular fractions of the sample before
analysis.
[0199] B) Sample Delivery into System.
[0200] The sample can be introduced into the devices of this
invention according to standard methods well known to those of
skill in the art. Thus, for example, the sample can be introduced
into the channel through an injection port such as those used in
high pressure liquid chromatography systems. In another embodiment
the sample can be applied to a sample well that communicates to the
channel. In still another embodiment the sample can be pumped into
the channel. Means of introducing samples into channels are well
known and standard in the capillary electrophoresis and
chromatography arts.
[0201] C) Sample Reaction with the Binding Agent.
[0202] The analyte containing sample is provided to the sensor
element in conditions compatible with or that facilitate binding of
the analyte to the binding agent comprising the sensor element.
Thus, for example, where the sensor element is an antibody or
protein, reaction conditions are provided at the sensor element
that facilitate antibody binding. Such reaction conditions are well
known to those of skill in the art (see, e.g., Techniques for using
and manipulating antibodies are found in Coligan (1991) Current
Protocols in Immunology Wiley/Greene, NY; Harlow and Lane (1989)
Antibodies: A Laboratory Manual Cold Spring Harbor Press, NY;
Stites et al. (eds.) Basic and Clinical Immunology (4th ed.) Lange
Medical Publications, Los Altos, Calif., and references cited
therein; Goding (1986) Monoclonal Antibodies: Principles and
Practice (2d ed.) Academic Press, New York, N.Y.; and Kohler and
Milstein (1975) Nature 256: 495-497, and the like).
[0203] Similarly, where the binding agent is a nucleic acid the
sensor element is maintained under conditions that facilitate
binding of the target nucleic acid (analyte) to the binding agent
comprising the sensor element(s). Stringency of the reaction can be
increased until the sensor shows adequate/desired specificity and
selectivity. Conditions suitable for nucleic acid hybridizations
are well known to those of skill in the art (see, e.g., Berger and
Kimmel, Guide to Molecular Cloning Techniques, Methods in
Enzymology 152 Academic Press, Inc., San Diego, Calif.; Sambrook et
al. (1989) Molecular Cloning--A Laboratory Manual (2nd ed.) Vol.
1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor Press, NY;
Ausubel et al. (1994) Current Protocols in Molecular Biology,
Current Protocols, a joint venture between Greene Publishing
Associates, Inc. and John Wiley & Sons, Inc.; U.S. Pat. No.
5,017,478; European Patent No. 0,246,864, and the like).
[0204] Once the analyte is bound to the binding agent in the sensor
element, the sensor is optionally dehydrated and/or stored and/or
read.
[0205] C) Analyte Detection/Quantitation.
[0206] Once introduced into the sensors of this invention, the
sample is detected/quantified using standard methods, e.g. as
described above, e.g. amperometry, voltammetry, coulometry, etc.
The measurement results can be compared to a standard curve, i.e. a
series or measurement results plotted as a function of analyte
concentration, which permits determination of analyte
concentration. The standard curve can be calculated by/stored in
the device performing data acquisition.
[0207] V. Cassettes.
[0208] In certain embodiments, this invention provides cassettes
comprising one or more sensor elements or sensor element arrays
according to this invention. In preferred embodiments, cassettes
include one or more biomolecules 14 and/or one or more working
electordes 10, 12 and/or biasing electrodes 22.
[0209] Thus, for example in certain embodiments, a cassette with
comprise a plurality of biomolecules 14, that are each attached to
a pair of electrodes. Counter electrodes are optionally provided,
e.g. integrated in the layer comprising the working electrodes or
provided as a component of a second layer comprising the
cassette.
[0210] In a preferred embodiment, a cassette or apparatus of the
invention comprises a sample port and/or reservoir and one or more
channels for sample delivery onto the sensor element(s) present in
the cassette. The means for sample delivery can be stationary or
movable and can be any known in the art, including but not limited
to one or more inlets, holes, pores, channels, pipes, microfluidic
guides (e.g., capillaries), tubes, and the like.
[0211] The channel(s) comprising the cassette of this invention can
comprise a channel network, e.g., one or more channels, preferably
microchannels. Typically included within a given channel network
are channels or reservoirs in which the desired analysis is to take
place (analysis channels), and thus the sensor elements of this
invention are disposed. Also, optionally included are channels for
delivering reagents, buffers, diluents, sample material and the
like to the analysis channels.
[0212] The cassettes of this invention optionally include
separation channels or matrices separating/fractionating materials
transported down the length of these channels, for analysis, i.e.,
size or charged based fractionation of materials, e.g., nucleic
acids, proteins etc. Suitable separation matrices include, e.g.,
GeneScan.TM. polymers (Perkin Elmer-Applied Biosystems Division,
Foster City, Calif.). Alternatively, analysis channels are devoid
of any separation matrix, and instead, merely provide a channel
within which an interaction, reaction etc., takes place. Examples
of microfluidic devices incorporating such analysis channels are
described in, e.g., PCT Application No. WO 98/00231, and U.S. Pat.
No. 5,976,336.
[0213] Fluids can be moved through the cassette channel system by a
variety of well known methods, for example: pumps, pipettes,
syringes, gravity flow, capillary action, wicking, electrophoresis,
electroosmosis, pressure, vacuum, etc. The means for fluid movement
may be located on the cassette or on a separate unit.
[0214] The sample can be placed on all of the sensor elements.
Alternatively, a sample may be placed on particular sensor
elements, e.g., by a capillary fluid transport means.
Alternatively, samples may be placed on the sensor element(s) by an
automatic pipetter for delivery of fluid samples directly to sensor
array, or into a reservoir in a cassette or cassette holder for
later delivery directly to the sensor element(s).
[0215] The cassettes of this invention can be fabricated from a
wide variety of materials including, but not limited to class,
plastic, ceramic, polymeric materials, elastomeric materials,
metals, carbon or carbon containing materials, alloys, composite
foils, silicon and/or layered materials. Supports may have a wide
variety of structural, chemical and/or optical properties. They may
be rigid or flexible, flat or deformed, transparent, translucent,
partially or fully reflective or opaque and may have composite
properties, regions with different properties, and may be a
composite of more than one material.
[0216] Reagents for conducting assays may be stored on the cassette
and/or in a separate container. Reagents can be stored in a dry
and/or wet state. In one embodiment, dry reagents in the cassette
are rehydrated by the addition of a test sample. In a different
embodiment, the reagents are stored in solution in "blister packs"
which are burst open due to pressure from a movable roller or
piston. The cassettes may contain a waste compartment or sponge for
the storage of liquid waste after completion of the assay. In one
embodiment, the cassette includes a device for preparation of the
biological sample to be tested. Thus, for example, a filter may be
included for removing cells from blood. In another example, the
cassette may include a device such as a precision capillary for the
metering of sample.
[0217] A cassette or apparatus of the invention can, optionally,
comprise reference electrodes, e.g., Ag/AgCl or a saturated calomel
electrode (SCE) and/or various biasing/counter-electrodes.
[0218] The cassette can also comprise more one layer of electrodes.
Thus, for example, different electrode sets (e.g. arrays of sensor
elements) can exist in different lamina of the cassette and thus
form a three dimensional array of sensor elements.
[0219] VI. Integrated Assay Device/Apparatus.
[0220] State-of-the-art chemical analysis systems for use in
chemical production, environmental analysis, medical diagnostics
and basic laboratory analysis are preferably capable of complete
automation. Such total analysis systems (TAS) (Fillipini et al.
(1991) J. Biotechnol. 18: 153; Garn et al (1989) Biotechnol.
Bioeng. 34: 423; Tshulena (1988) Phys. Scr. T23: 293; Edmonds
(1985) Trends Anal. Chem. 4: 220; Stinshoff et al. (1985) Anal.
Chem. 57:114R; Guibault (1983) Anal. Chem Symp. Ser. 17: 637;
Widmer (1983) Trends Anal. Chem. 2: 8) automatically perform
functions ranging from introduction of sample into the system,
transport of the sample through the system, sample preparation,
separation, purification and detection, including data acquisition
and evaluation.
[0221] Recently, sample preparation technologies have been
successfully reduced to miniaturized formats. Thus, for example,
gas chromatography (Widmer et al. (1984) Int. J. Environ. Anal.
Chem. 18: 1), high pressure liquid chromatography (Muller et al.
(1991) J. High Resolut. Chromatogr. 14: 174; Manz et al. (1990)
Sensors & Actuators B1:249; Novotny et al., eds. (1985)
Microcolumn Separations: Columns, Instrumentation and Ancillary
Techniques J. Chromatogr. Library, Vol. 30; Kucera, ed. (1984)
Micro-Column High Performance Liquid Chromatography, Elsevier,
Amsterdam; Scott, ed. (1984) Small Bore Liquid Chromatography
Columns: Their Properties and Uses, Wiley, N.Y.; Jorgenson et al.
(1983) J. Chromatogr. 255: 335; Knox et al. (1979) J. Chromatogr.
186:405; Tsuda et al. (1978) Anal. Chem. 50: 632) and capillary
electrophoresis (Manz et al. (1992) J. Chromatogr. 593: 253;
Olefirowicz et al. (1990) Anal. Chem. 62:1872; Second Int'l Symp.
High-Perf. Capillary Electrophoresis (1990) J. Chromatogr. 516;
Ghowsi et al. (1990) Anal. Chem. 62:2714) have been reduced to
miniaturized formats.
[0222] Similarly, in certain embodiments, this invention provides
an integrated assay device (e.g., a TAS) for detecting and/or
quantifying one or more analytes using the sensor elements, sensor
element arrays, or cassettes of this invention.
[0223] Thus, in certain embodiments, the cassettes of this
invention are designed to be inserted into an apparatus, that
contains means for reading one or more sensor elements comprising a
cassette of this invention. The apparatus, optionally includes
means for applying one or more test samples onto the sensor
elements or into a receiving port or reservoir and initiating
detecting/quantifying one or more analytes. Such apparatus may be
derived from conventional apparatus suitably modified according to
the invention to conduct a plurality of assays based on a support
or cassette. Modifications required include the provision for,
optional, sample and/or cassette handling, multiple sample
delivery, multiple electrode reading by a suitable detector, and
signal acquisition and processing means.
[0224] Preferred apparatus, in accordance with this invention, thus
typically include instrumentation suitable for performing
electrochemical measurements and associated data acquisition and
subsequent data analysis.
[0225] Preferred apparatus also provide means to hold cassettes,
optionally provide reagents and/or buffers and to provide
conditions compatible with binding agent/target analyte binding
reactions.
[0226] A preferred apparatus also comprises an electrode contact
means able to electrically connect the array of separately
addressable electrode connections of the cassette to an
electronic-voltage/waveform generator, e.g., potentiostat. The
waveform generator means delivers signals sequentially or
simultaneously to independently read a plurality of sensor elements
in the cassette.
[0227] The apparatus optionally comprises a digital computer or
microprocessor to control the functions of the various components
of the apparatus.
[0228] The apparatus also comprises signal processing means. In one
embodiment, and simply by way of example, the signal processing
means comprises a digital computer for transferring, recording,
analyzing and/or displaying the results of each assay.
[0229] The sensor element arrays of this invention are particularly
well suited for use as detectors in "low sample volume"
instrumentation. Such applications include, but are not limited to
genomic applications such as monitoring gene expression in plants
or animals, parallel gene expression studies, high throughput
screening, clinical diagnosis, single nucleotide polymorphism (SNP)
screening, genotyping, and the like. Certain particularly preferred
embodiments, include miniaturized molecular assay systems,
so-called labs-on-a-chip, that are capable of performing thousands
of analyses simultaneously
[0230] Kits.
[0231] In certain embodiments, this invention provides kits for
practice of the methods and/or assembly of the devices described
herein. Preferred kits comprise a container containing one or more
sensor elements according to the present invention. The sensor
elements can be components of a sensor array and/or can comprise a
sensor cassette as describe herein. In certain embodiments, the
kits, optionally, include one or more reagents and/or buffers for
use with the sensors of this invention. The kits can optionally
include materials for sample acquisition, processing, and the
like.
[0232] The kits can also include instructional materials containing
directions (i.e., protocols) for the practice of the assay methods
of this invention the use of the cassettes described herein,
methods of assembling sensor elements into various instruments, and
the like. While the instructional materials typically comprise
written or printed materials they are not limited to such. Any
medium capable of storing such instructions and communicating them
to an end user is contemplated by this invention. Such media
include, but are not limited to electronic storage media (e.g.,
magnetic discs, tapes, cartridges, chips), optical media (e.g., CD
ROM), and the like. Such media may include addresses to internet
sites that provide such instructional materials.
EXAMPLES
[0233] The following examples are offered to illustrate, but not to
limit the claimed invention.
Example 1
[0234] Sensor Element Formation.
[0235] Alternating layers of insulators and conductors are formed
by sputtering or vapor deposition (e.g. as described in U.S. Pat.
No. 5,414,588. The layers consist of a substrate (Alkali-free
borosilicate glass (Shott AF45)), followed by a first conductor,
then an insulator, followed by a second conductor and so forth. The
first conductor plus insulator, and the second conductor plus a
second insulator comprise one iteration. Iterations are repeated
until the desired number of lamina is achieved.
[0236] The position of the conductors and insulators is determined
by a mask. Thus, as illustrated in FIG. 24A, conductor 1 has a
designated mask and/or mask position of the mask determining the
location of its deposition. Similarly, the insulator position is
determined by the use of a second mask, as illustrated in FIG. 24B,
and the position of conductor 2 is determined by a third mask as
illustrated in FIG. 24C. The masks are reused for each subsequent
iteration for a total of ten iterations.
[0237] The sputtering process results in a multi-laminar structure
of alternating conducting and insulating layers where the first
conductor layers are connected to each other and the second layers
to be connected to each other, but not to the first conductor
layers (see FIG. 24D) similar to the capacitor described in U.S.
Pat. No. 5,414,588.
[0238] The conductors are fabricated of gold, and the insulator
layers are made of glass or polystyrene or teflon.
[0239] The multilayer structure is cut to expose the thin layers of
conductors and insulators. The exposed surface is then polished
smooth. In selected structures, the insulator layers are etched
further to form a channel between the conductive layers.
[0240] The first conductor layers are connected to a first
macro-electrode using common semi-conductor etching methods. The
second conductor layers are connected to a second macro-electrode
also using common semi-conductor etching methods.
[0241] The macro-electrodes are connected to a voltage source and
tested for non-conductance using an EG&G High Speed
Potentiostat/Galvanostat (PerkinElmer Model 283).
[0242] Analyte Detection.
[0243] The multilayer electrode face is contacted with a capture
probe solution comprising 30 mer oligonucleotides. The 5 prime end
of the oligonucleotides is derivatized with an electrolabile, an
alkyl- or aryl chloroformate, which can be removed at -1.5 volts in
the presence of LiClO.sub.4/CH.sub.3OH to reveal a thiol group
which can then form a covalent bond with a gold electrode.
[0244] The 3 prime end of the oligonucleotide is derivatized with
another electrolabile group such as S-benzyloxycarbonyl moiety
which can removed at -2.6 volts in DMF and tetrabutyl ammonium
chloride. Each of the electrolabile groups is cleaveable at a
unique voltage.
[0245] The first conductor is biased with the activation voltage of
the 5 prime electrolabile group on the capture probe thereby
exposing the thiol group which then attaches to the first
conductor.
[0246] The solution comprising the analyte (a nucleic acid
comprising a sequence complementary to the capture probe) is
contacted with the capture probe and allowed to hybridize to the
capture probe on the electrodes.
[0247] The second conductor is biased with the activation voltage
of the 3 prime electrolabile group of the capture probe thereby
attaching the probe to connect to the second conductor. The
electrodes are then dried under nitrogen or argon.
[0248] The electrodes are connected to a macro electrodes to a
voltage source and tested for non-conductance, or a background
conductance, is measured using a high-speed
potentiostat/galvanostat (e.g. Perkin-Elmer, Model 283).
[0249] The electrodes are dried again under nitrogen or argon. A
voltage (e.g., -6-+6V,) is applied again to the electrodes and the
current is measured. The measured current of the hybridized nucleic
acids is significantly greater the current measured for the
unhybridized electrodes.
[0250] It is understood that the examples and embodiments described
herein are for illustrative purposes only and that various
modifications or changes in light thereof will be suggested to
persons skilled in the art and are to be included within the spirit
and purview of this application and scope of the appended claims.
All publications, patents, and patent applications cited herein are
hereby incorporated by reference in their entirety for all
purposes.
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